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Departments of Obstetrics and Gynecology (J.-J.L., M.-C.O.-C.), Biochemistry (W.-L.Z., M.E.N., D.E.O.), Cell Biology (G.-Q.Z., S.K., R.J.M., M.-C.O.-C.), Urologic Surgery (S.K., R.J.M.), and Center for Reproductive Biology Research (S.K., M.E.N., R.J.M., D.E.O., M.-C.O.-C.), Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-2633
Address all correspondence and requests for reprints to: Marie-Claire Orgebin-Crist, Center for Reproductive Biology Research, Vanderbilt University, School of Medicine, Medical Center North, Room D2303, Nashville, Tennessee 37232-2633.
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Introduction |
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Protein secretion by the male sex accessory organs is mainly under androgenic control (3, for review) but unidentified testicular factors (4), estrogens (5), and retinoic acid (6) have also been implicated. An interesting feature of epididymal protein secretion is that, although the epididymis is composed of one primary cell type throughout its length, specific gene expression is highly regionalized (3, for review).
We previously described two peptides (major and minor forms) resulting from a differential cleavage of a unique precursor that we named MEP 10 (mouse epididymal protein 10) (2). This protein is synthesized by principal cells of the distal caput epididymidis and is secreted into the lumen. MEP 10 is not tightly bound to spermatozoa because it can be easily dissociated under low-salt conditions (2). N-terminal microsequencing revealed a high homology (86%) with two rat epididymal secretory proteins (7) that were later identified as epididymal retinoic acid-binding proteins (8, 9). MEP 10 binds active retinoids (cis and all-trans retinoic acid) but not retinol (10). The gene encoding the rat epididymal retinoic acid-binding proteins has been characterized and shown to belong to the lipocalin superfamily (11). These rat epididymal secretory proteins were previously named proteins B/C (7, 12), epididymal binding proteins (EBP l and 2) (8), ESP I (11), and E-RABP (13, 14). To conform to the international nomenclature, we propose the generic term "E-RABP" (epididymal retinoic acid-binding protein) to name these proteins. MEP 10 will be referred to as mE-RABP (murine epididymal retinoic acid-binding protein).
In this report, we describe the molecular cloning of a complementary DNA (cDNA) encoding the mature form of mE-RABP. We show that mE-RABP is a new member of the lipocalin superfamily and the mouse orthologue protein of the rat epididymal retinoic acid-binding protein (ESP I). In addition, we demonstrate that the tissue-specific expression and the hormonal regulation of the mE-RABP gene differs in the rat and the mouse.
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Materials and Methods |
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Isolation of the cDNA encoding the mature form of mE-RABP
Total RNA was extracted from the epididymis as described
previously (15). For RT, an aliquot (2 µg) was incubated in a mixture
containing 50 mM Tris-HCI (pH 8.3), 75 mM KCl,
10 mM DTT, 3 mM MgCl2, 0.5
mM each dGTP, dATP, dCTP, dTTP, 2 U/µl RNAsin (Promega,
Madison, WI), 10 µg/ml oligonucleotide RACEIII
(5'-CAGCTGCAGGTACCGGATCCTCGAGAAGC(T)18-3', and 200 U
Moloney murine leukemia virus reverse transcriptase (MoMLV) for 1
h at 42 C. The DNA/RNA hybrids were denatured for 5 min at 90 C and
stored at -80 C. Then, a PCR reaction was performed using 100 ng of
single strand cDNA incubated in a mixture containing 0.2
µM each dGTP, dATP, dTTP, dCTP, 1 µM each
primers (FwE-RABP: 5'-GCGAATTCACCATGGCNGTNGTNAARGAYTTYGAY-3' and RiIII:
5'-GCAGGTACCGGATCCTCGAGAAG-3'), 1 x reaction buffer II (Perkin
Elmer, Foster City, CA), and 1 U AmpliTaq (Perkin Elmer). DNA was
amplified for 25 cycles, each consisting of 1 min at 94 C, 2 min at 55
C, and 2 min at 72 C. Amplified fragments were purified on a 2%
(wt/vol) agarose gel, digested with EcoRl and
BamHl restriction enzymes (Promega), and ligated into
pGEM7Zf(-) plasmid (Promega). DNA sequencing was performed as described
in the thermo sequenase fluorescent labeled primer cycle sequencing kit
(Perkin Elmer).
In vitro transcription/translation assays
Sense or antisense RNA were transcribed from the E7 clone using
the T7 or the SP6 RNA polymerase, respectively. All transcription and
translation reactions were performed according to the manufacturer’s
instructions (Promega). Briefly, 5 µg of linearized E7 plasmid were
incubated with 10 mM dithiothreitol (DTT), 5 U RNAzin RNAse
inhibitor (Promega), 0.5 mM rNTP, 1 x enzyme buffer,
and 5 U of the appropriate RNA polymerase for 1 h at 37 C.
Translation of 5 µg of RNA was carried out in 50 µl containing 35
µl of rabbit reticulocyte lysate (Promega), 50 µCi (1200 Ci/mmol)
[35S] methionine (Amersham, Arlington Heights, IL), 50 U
RNAzin RNAse inhibitor (Promega), and a 20 µM amino acid
mixture (minus methionine) for 2 h at 30 C. Translation products
were immunoprecipitated as described previously (16) using 20 µg of
purified preimmune or immune IgG (2). Samples (106 cpm)
were treated and analyzed by one-dimensional SDS-PAGE [15% (wt/vol)]
acrylamide, 0.1% (wt/vol) bisacrylamide) (17).
In situ hybridization
The epididymis was fixed overnight in freshly prepared 4%
(vol/vol) paraformaldehyde-PBS (pH 7.4), rinsed with PBS and then
dehydrated in a series of increasing concentrations of ethanol for a
period of 3–5 h. After being embedded in paraplast, tissues were
sectioned at 7 µm. RNA probes were labeled with [35S]
UTP to a specific activity of l.2 x l09 cpm/µg.
Hybridization was carried out at 50 C with 2 x 104
cpm/µl riboprobe overnight in 50% (vol/vol) formamide, 300
mM NaCl, 10 mM Tris (pH 7.4), 10 mM
NaH2PO4 (pH 6.8), 5 mM EDTA (pH 8),
0.2% (wt/vol) Ficoll 400, 0.2% (wt/vol) polyvinylpyrrolidone, 10%
(wt/vol) dextran sulfate, 200 µg/ml yeast transfer RNA, and 50
mM DTT. Excess of riboprobe was eliminated by two
high-stringency washes in 2 x SSC, 50% (vol/vol) formamide at 65
C. Slides were dipped in NTB-2 Kodak emulsion and exposed for 24 h
at 4 C, developed, fixed, and mounted with Permount (Fisher, NJ) for
photography.
Northern blotting
Total RNA was extracted as described previously (15). Total RNA
(10 µg) was denatured 15 min at 65 C and cooled on ice. RNA samples
were loaded on a 1% (wt/vol) agarose gel containing 20 mM
3-[N-morpholino]propanesulfonic acid (MOPS) (pH 7), 5
mM sodium acetate, 1 mM EDTA, 6% (vol/vol)
formaldehyde, and then transferred to a Hybond N+ nylon
membrane (Amersham) by blotting overnight in 20 x SSC. The
membrane was washed once in 2 x SSC, dried, and baked 2 h at
80 C. The prehybridization was carried out for 3 h at 42 C in 50%
(vol/vol) formamide, 6 x SSC, 5% (vol/vol) Denhardt’s, 100
µg/ml salmon sperm DNA, 0.1% (wt/vol) SDS, and then random primed
[32P]-labeled mE-RABP cDNA, prepared with "Random
prime" kit (Amersham), was added and incubated overnight. The filter
was washed once in 2 x SSC for 15 min, once in 2 x SSC,
0.1% (wt/vol) SDS for 15 min, once in 2 x SSC, 0.1% (wt/vol)
SDS for 30 min, once in 0.2 x SSC, 0.1% (wt/vol) SDS for 15 min,
and once in 0.l x SSC, 0.1% (wt/vol) SDS for 15 min at 65 C
before being autoradiographed with a Hyperfilm MP (Amersham). Northern
blots were reprobed with a cloned 18S cDNA to standardize the loaded
RNA samples. The relative absorbance of the mE-RABP and 18S RNA was
determined using an imaging densitometer (model GS-670, Bio-Rad,
Hercules, CA) and the Multi Analyst software (Bio-Rad).
Western blotting
Tissues were homogeneized in 10 mM Tris-HCl, pH 7.4,
150 mM NaCl in the presence of protease inhibitors
(leupeptin 1 µg/ml, chymostatin 1 µg/ml, aprotinin 1 µg/ml,
antipain 2 µg/ml, benzamidine 10 µg/ml). Samples were centrifuged
at 40,000 x g, and the supernatants were stored at
-80 C before use. Twenty micrograms of total protein were separated on
a SDS-PAGE (17% polyacrylamide gel) and transferred to a Protran
nitrocellulose membrane as described previously (18). Nitrocellulose
membranes were incubated overnight at 4 C in PBS/1% (wt/vol) BSA,
washed five times in PBS/0.1% (wt/vol) BSA, 0.1% (vol/vol) Tween-20,
incubated for 1 h with the immune rabbit IgG anti-mE-RABP (2),
washed, incubated for 1 h at room temperature with biotinylated
antirabbit IgG ("Vectastain kit", Vector Laboratories, Inc.),
washed, and incubated for 1 h at room temperature in the
ABC-peroxidase reagent (Vector Laboratories, Inc.). After washing five
times in PBS/0.1% (vol/vol) Tween-20 and once in PBS 1x, reactive
bands were visualized using a solution containing 0.5 mg/ml
diaminobenzidine (DAB), 0.02% H2O2, 0.04%
NiCl2 in 0.05 M Tris-HCl, pH 7.5. The reaction
was stopped with H2O, and the membrane was air dried. The
relative absorbance of mE-RABP was determined using an imaging
densitometer (model GS-670, Bio-Rad) and the Multi Analyst
software.
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Results |
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).
The first thirteen amino acids at the N-terminal end of the peptide
were identical to the published microsequence of the minor form of
mE-RABP (2). To confirm that this peptide was immunologically related
to mE-RABP, in vitro transcription/translation assays were
carried out (Fig. 2). The sense RNA
transcribed from the T7 RNA polymerase promoter encoded an 18 kDa
peptide which was specifically immunoprecipitated with a rabbit
polyclonal antibody raised against mE-RABP (2). This antibody was
unable to immunoprecipitate any peptide produced in presence of
antisense RNA. Altogether, these results demonstrate that the clone E7
containing the 0.6 kb DNA fragment encoded the minor form of
mE-RABP.
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). The highest match
(75% identity, 90% homology) was with rat ESP I which is also
specifically expressed in the epididymis and is under androgen control
(11). In addition, mE-RABP and ESP I have the same hydropathic pattern
(Fig. 4). Other members of the lipocalin
family, including mouse major urinary protein (19), rat retinol binding
protein (20), sheep ß-Lactoglobulin (21), 
-1-acid glycoprotein
(22) and lizard epididymal secretory protein IV (23), showed
significant but lower homologies (26.5% identity, 48% similarity)
with mE-RABP (Fig. 3).
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). Only three changes were found in the
retinoic acid binding cavity, which encompasses 23 amino acids (Fig. 5B). In the mouse protein, the conversion of the residues Ile to Val at
position 8 (rIle-8/mVal), Phe to Tyr at position 76 (rPhe-76/mTyr) and
Leu to Tyr at position 50 (rLeu-50/mThr) results in a binding cavity
only slightly larger than that described for rat E-RABP, but it still
conforms well to the contours of the ligand. Thus, mE-RABP is the mouse
equivalent of rat ESP I.
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). The region-specific expression of
mE-RABP mRNA was first studied by Northern blot analyses (Fig. 6B).
mE-RABP mRNA was detected in the caput but not in the corpus or the
cauda. In situ hybridization experiments showed the highly
restricted expression of mE-RABP mRNA within the caput (Fig. 7). No expression was detected in the
efferent ducts and initial segment (1). A low level of expression,
detectable only by dark field microscopy, was seen in segment 2
[classification of Abou-Haila and Fain-Maurel (24)]. Expression
progressively increased in segment 3, reaching a maximum in the distal
portion of segment 3 and segment 4. mE-RABP mRNA expression abruptly
decreased in segment 5 where, interestingly, a checkerboard pattern was
observed, i.e. some principal cells expressed mE-RABP mRNA
and others did not. No expression was seen in the corpus, the cauda or
the vas deferens.
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).
The steady state level of mE-RABP mRNA decreased to 11% of control
intact animals by 5 days after castration and became almost
undetectable (0.7%) by 10 days. However, a rebound from 0.7% to 5%
of the mE-RABP mRNA was noted in mice castrated for 15 days. The
mE-RABP mRNA expression increased progressively from day 15 to day 60
when it reached 18% of the control level. Western blot analysis showed
that mE-RABP was still present 5 days after castration (0.5% of
control) in the epididymis but was not detectable 20 days after
castration despite a similar level of mE-RABP mRNA being present 5 days
and 20 days after castration (11% and 13.6% of control,
respectively). Therefore, the rebound of the mE-RABP mRNA did not lead
to a recovery of the protein.
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In situ hybridization was used to determine the localization
of the mE-RABP mRNA after androgen withdrawal and testosterone
replacement (Fig. 9 and 10). mE-RABP mRNA expression was not
detectable 45 days after castration due to the limited time of film
exposure (1 day). However, injections of testosterone propionate for 10
days to 45-day castrated animals restored mE-RABP mRNA expression.
Although testosterone replacement did not restore the size of the
organ, a similar segment-specific expression of mE-RABP mRNA to that
seen in the intact animal was observed. The major differences were a
loss of the checkerboard pattern of mE-RABP expression at the junction
of the caput and the corpus seen in the intact animal and expression in
the proximal corpus while in the intact animal there was an abrupt
disappearance of expression in the corpus (Fig. 10). Altogether, these
results indicate that the steady state level and region-specific
expression of the mE-RABP mRNA are not dependent on testicular
factor(s) present in the luminal fluid but are dependent on testicular
androgens present in the blood.
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Discussion |
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).
Analysis of the derived primary amino acid sequence revealed that
mE-RABP was a new member of the lipocalin superfamily (Fig. 3). This
family includes a large number of secretory proteins (26, for review),
but statistical analysis showed that the identity between any pair may
be as low as 20–30%. Despite the low overall amino acid sequence
identity (26.5% identity, 48% similarity), mE-RABP contains two
motifs Gly-X-Trp and Thr-Asp-Tyr as well as two cysteine residues that
are highly conserved among the members of the lipocalin family (27) and
are believed to be important for the three-dimensional structure of
lipocalins. Indeed, all available crystal structures show the
lipocalins to have a similar three dimensional structure (human plasma
retinol binding protein (RBP) (28), human ß-lactoglobulin (Blg) (29),
insecticyanin (30), bilin-binding protein (BBP) (31), mouse major
urinary protein (MUP) and rat 2u-globulin (32), bovine
odorant-binding protein (OBP) (33, 34), and rat epididymal retinoic
acid binding protein (E-RABP) (13). All lipocalins have an eight
stranded up-and-down ß-barrel closed at one end by a single turn of
the -helix and a hydrophobic binding cavity. The ligand binding
domain is well adapted for noncovalent binding of small hydrophobic
ligands (retinol, retinoic acid, biliverdin, porphyrin, cholesterol
derivates, pheromones, or prostaglandins) (26, for review).
The mE-RABP had the highest amino acid sequence identity (75%) with a
rat androgen-regulated epididymal secretory protein named protein B/C
(7), also known as EBP (8, 9), E-RABP (13, 14), and ESP I (11). This
protein, identified in the luminal fluid of the rat epididymis (7, 12),
is a retinoic acid binding protein (8) encoded by a single-copy gene
(11). Rat E-RABP, like mE-RABP, binds both all-trans and
9-cis retinoic acid (13) but not retinol (8). A comparison
of the putative mE-RABP three-dimensional structure to that of ESP I
showed that some amino acids in the ligand binding domain were
different. However, these minor changes do not affect the space
required to bind retinoic acid (Fig. 5). The amino acid sequence
homology, the ligand-binding specificity, and the epididymis-specific
expression of a single-copy gene (35) confirm that mE-RABP is the mouse
orthologue of the rat epididymal secretory protein.
Another androgen-dependent lipocalin related to mE-RABP (26.5% identity, 48% similarity) was described in the lizard epididymal fluid (23, 36). This suggests that an E-RABP related to the lipocalin superfamily is conserved during evolution and may have an important function in male fertility.
Tissue-and region-specific expression of the mE-RABP gene
Although the epididymis consists of a single convoluted duct
composed of one major cell type, it displays a high degree of
region-specific expression of a large number of genes (3, for review).
Northern blot analyses have shown that mE-RABP mRNA expression is
restricted to the caput epididymidis (Fig. 8). This observation is in
agreement with previous studies showing that mE-RABP was synthesized
and secreted into the luminal fluid from the mid/distal caput
epididymidis (2, 37, 38). mE-RABP was also found by
immunohistochemistry in the cytoplasm of clear cells in the corpus and
cauda epididymidis (2). In situ hybridization showed that
mE-RABP mRNA was gradually expressed in the principal cells from the
proximal to the distal caput epididymidis (Fig. 7). However, no mE-RABP
mRNA was detected in clear cells located in the corpus and cauda
epididymidis (data not shown). This result is in agreement with our
previous report suggesting that clear cells endocytosed mE-RABP from
the luminal fluid (38). The mE-RABP is related to retinol binding
protein that binds retinol and forms a complex with another plasma
protein named Transtherithin (39). This complex is able to deliver
retinol to cells expressing a specific receptor on their surface (40).
Therefore, it is reasonable to expect that a mE-RABP-specific receptor
exists on clear cell plasma membranes allowing the endocytosis of
mE-RABP from the luminal fluid.
The increase in mE-RABP mRNA expression in the caput was gradual within all principal cells. In contrast, the disappearance of expression at the junction of the caput and the corpus was abrupt, but some principal cells continued to exhibit strong expression, whereas adjacent cells display none, giving a checkerboard appearance to segment 5. Such a cell-specific gene expression has been observed for several epididymal mRNA and/or proteins (4, for review).
Although mE-RABP and rat proteins B/C are homologous, the mRNA
expression patterns are different. In the rat, proteins B/C mRNA are
expressed uniformly in the efferent duct, and in the initial, proximal
and distal caput epididymidis (41). In the mouse, mE-RABP mRNA is
expressed in a gradient pattern from the mid to the distal caput
epididymidis but is undetectable in the efferent ducts and the initial
segment (Fig. 7). Although it is difficult to compare the segments of
the caput epididymidis from different species, our result suggests that
the regulation of region-specific expression of E-RABP may be different
in mouse and rat epididymis.
Androgen regulation of the mE-RABP gene expression
Androgens are required for epididymal sperm maturation (42, 43).
Previous studies have shown that castration of adult mice results in
the loss of the mE-RABP expression (37). Androgens could regulate
mE-RABP translation or stability, but Northern blot analyses showed
that castration, but not efferent duct ligation, led to a rapid
decrease of mE-RABP mRNA expression. Moreover, testosterone propionate
or DHT injections to castrated mice restored mE-RABP mRNA expression.
These results suggest that mE-RABP mRNA expression is dependent only on
circulating androgens. When mE-RABP expression reappeared after
androgen replacement, the same overall region-specific pattern of
expression seen in the intact mice was observed. However, the
transition between the caput and the corpus was not as abrupt and
mE-RABP mRNA was expressed in the proximal corpus region. The
checkerboard pattern of expression seen in the intact animal was not
present, and all principal cells expressed similar levels of mRNA. This
suggests that the factors involved in the region-specific and
cell-specific expression of mE-RABP may be affected by castration and
testosterone replacement.
Androgens bind to a nuclear receptor that belongs to the ligand-inducible transcription factors superfamily (44). The binding of the androgen receptor to specific cis-acting DNA regulatory elements named the androgen response element (ARE) results in the modulation of the target gene transcription (45). A nucleotide sequence homologue to this regulatory element is present in the rat ESP I gene promoter (11). Therefore, mE-RABP gene expression may be directly regulated by androgens at the transcriptional level. However, in the prostate, androgens regulate mitochondrial aspartate aminotransferase (mAAT) (46) and prostatic binding protein (PBP) C3(1) gene expression (47) primarily by controlling the stability of the mRNA. Thus, androgens may also regulate the turn over of mE-RABP mRNA.
mE-RABP mRNA expression progressively reappeared between 15 and 60 days
after castration (Fig. 8). In the prostate, an androgen-dependent mRNA
encoding another lipocalin protein named probasin was observed to
rebound to precastrated levels 12 days after castration (48). However,
to our knowledge, the rebound of an androgen-regulated gene never has
been reported before in the epididymis. Moreover, rat proteins B/C gene
expression does not rebound after long-term castration (49), suggesting
that this rebound of mE-RABP is unique to the mouse. It is unlikely
that adrenal androgens are involved because the level of epididymal
androgens stay low during long term castration in the mouse (25). This
atypical behavior suggests that other transcription factor(s)
(repressor or activator) may cooperate with the androgen receptor to
control mE-RABP gene expression at a transcriptional and/or
posttranscriptional level. Further studies will be required to
elucidate this phenomenon.
At 5 days, but not at 30 days post castration, a significant amount of mE-RABP protein was present in the epididymis despite a similar level of mE-RABP mRNA. This suggests that the synthesis or the stability of mE-RABP protein, in vivo, may be dependent on testicular factor(s) and/or androgens. Such posttranscriptional regulation has been described for other epididymal genes (25, 50). For example, the transcription and the steady-state levels of the GPX5 mRNA are fully restored after castration and testosterone replacement but not the translation of the GPX5 mRNA suggesting that testicular factors exert a posttranscriptional control for GPX5 synthesis (25).
In summary, mE-RABP belongs to the lipocalin superfamily. Because the lipocalins are mainly extracellular transport proteins involved in the delivery of small lipophilic molecules (26), the most plausible function for mE-RABP is as transport protein for retinoic acid within the epididymis. The conservation of this protein during evolution suggests that it has an important function in male fertility.
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Acknowledgments |
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Received December 11, 1997.
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N. M. Ibrahim, L. G. Young, and O. Frohlich Epididymal Specificity and Androgen Regulation of Rat EP2 Biol Reprod, August 1, 2001; 65(2): 575 - 580. [Abstract] [Full Text] [PDF]
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M. Govoroun, O. M. McMeel, H. Mecherouki, T. J. Smith, and Y. Guiguen 17{beta}-Estradiol Treatment Decreases Steroidogenic Enzyme Messenger Ribonucleic Acid Levels in the Rainbow Trout Testis Endocrinology, May 1, 2001; 142(5): 1841 - 1848. [Abstract] [Full Text]
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P. P. Lahti, R. Shariatmadari, J. K. Penttinen, J. R. Drevet, B. Haendler, M. Vierula, M. Parvinen, I. T. Huhtaniemi, and M. Poutanen Evaluation of the 5'-Flanking Regions of Murine Glutathione Peroxidase Five and Cysteine-Rich Secretory Protein-1 Genes for Directing Transgene Expression in Mouse Epididymis Biol Reprod, April 1, 2001; 64(4): 1115 - 1121. [Abstract] [Full Text]
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J.-J. Lareyre, V. P. Winfrey, S. Kasper, D. E. Ong, R. J. Matusik, G. E. Olson, and M.-C. Orgebin-Crist Gene Duplication Gives Rise to a New 17-Kilodalton Lipocalin That Shows Epididymal Region-Specific Expression and Testicular Factor(s) Regulation Endocrinology, March 1, 2001; 142(3): 1296 - 1308. [Abstract] [Full Text] [PDF]
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J.-J. Lareyre, K. Reid, C. Nelson, S. Kasper, P. S. Rennie, M.-C. Orgebin-Crist, and R. J. Matusik Characterization of an Androgen-Specific Response Region Within the 5' Flanking Region of the Murine Epididymal Retinoic Acid Binding Protein Gene Biol Reprod, December 1, 2000; 63(6): 1881 - 1892. [Abstract] [Full Text]
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 E. Lacazette, A.-M. Gachon, and G. Pitiot A novel human odorant-binding protein gene family resulting from genomic duplicons at 9q34: differential expression in the oral and genital spheres Hum. Mol. Genet., January 22, 2000; 9(2): 289 - 301. [Abstract] [Full Text] [PDF]
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 J.-J. Lareyre, T. Z. Thomas, W.-L. Zheng, S. Kasper, D. E. Ong, M.-C. Orgebin-Crist, and R. J. Matusik A 5-Kilobase Pair Promoter Fragment of the Murine Epididymal Retinoic Acid-binding Protein Gene Drives the Tissue-specific, Cell-specific, and Androgen-regulated Expression of a Foreign Gene in the Epididymis of Transgenic Mice J. Biol. Chem., March 19, 1999; 274(12): 8282 - 8290. [Abstract] [Full Text] [PDF]
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