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5-4 Isomerase Gene: Activation by Prolactin
Vanderbilt University School of Medicine (F.A.F., M.H.M.)
Departments of Obstetrics/Gynecology and Cell Biology Nashville,
Tennessee 37232
Institute for Biomedical Research (B.G.)
Georg-Speyer-Haus Frankfurt, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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5-4 isomerase (3ß-HSD). A
9/9 match with the consensus Stat5 response element was identified at
-110 to -118 in the human Type II 3ß-HSD promoter. 3ß-HSD
chloramphenicol acetyltransferase (CAT) reporter constructs containing
either an intact or mutated Stat5 element were tested for PRL
activation. Expression vectors for Stat5 and the PRL receptor were
cotransfected with a -300 
+45 3ß-HSD CAT reporter construct into
HeLa cells, which resulted in a 21-fold increase in reporter activity
in the presence of PRL. Promoter activity showed an increased response
with a stepwise elevation of transfected Stat5 expression or by
treatment with increasing concentrations of PRL (max, 250 ng/ml). This
effect was dramatically reduced when the putative Stat5
response element was removed by 5'-deletion of the promoter or by
the introduction of a 3-bp mutation into critical nucleotides in
the element. Furthermore, 32P-labeled
promoter fragments containing the Stat5 element were shifted in
electrophoretic mobility shift assay experiments using nuclear
extracts from cells treated with PRL, and this complex was supershifted
with antibodies to Stat5. These results demonstrate that PRL has the
ability to regulate expression of a key human enzyme gene (type II
3ß-HSD) in the progesterone biosynthetic pathway, which is essential
for maintaining pregnancy. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The role of PRL in human reproduction is evident when circulating PRL is dramatically altered from normal physiological levels. Hypoprolactinemia induced by bromocriptine treatment of normal women has been shown to affect the length of the luteal cycle and circulating levels of progesterone (7). Conversely, hyperprolactinemia is associated with infertility in women (8), and elevated serum PRL levels in these patients may result in galactorrhea and amenorrhea. Functional targets of PRL in the human reproductive tissues are unclear, but a putative site of action is the ovary with effects on folliculogenesis and corpus luteum (CL) function.
PRL effects are mediated by members of the PRL/placental lactogen (PL) family in the rat and PRL/GH/PL family in humans (9). Tissue-specific effects of PRL and PRL-like molecules are regulated by cell surface expression of PRL receptors. The PRL receptor is a single-pass transmembrane receptor of the cytokine receptor superfamily (10), and it is alternatively transcribed from a single gene resulting in expression of at least two isoforms: long and short (11). The long form of the PRL receptor has been shown to transduce PRL signals primarily by activation of Stat5 through the Jak/Stat pathway (12). PRL activation of Stat5 is thought to occur by ligand-dependent activation of the tyrosine kinase, Jak2, resulting in recruitment of latent Stat5 molecules via SH2 domains from the cytoplasm to the receptor complex. Jak2 subsequently phosphorylates Stat5 on tyrosine 694 (13), and the Stat5 molecules then dimerize via association with SH2 domains, translocate to the nucleus, and bind to Stat5 response elements in the regulatory regions of target genes, thereby activating transcription.
Some PRL-regulated genes in the rat ovary have been identified
including several genes involved in ovulation such as
2-macroglobulin (14), LH receptor (15), tissue
plasminogen activator (16), and plasminogen activator inhibitor type-1
(PAI-1) (16). PRL also appears to regulate genes encoding enzymes
involved with progesterone synthesis and metabolism in the rat CL
including 20-HSD (17), P450scc (18), and 3ß-HSD (19).
These examples demonstrate the broad scope of PRL action in the rat
CL.
While PRL/placental lactogens play a primary luteotrophic role in rodents, the function of PRL in conjunction with hCG in primates is less defined. Although PRL receptors have been demonstrated in the human ovary (20, 21), functional consequences of this binding have not been fully explored. Owing to the luteotrophic nature of PRL in the rat CL, it is possible that PRL might play a contributing role in the primate CL. PRL has been shown to increase basal progesterone production in antral follicles (22), dispersed corpora luteal cells (23), and in granulosa-lutein cell cultures obtained from women undergoing egg retrieval in in vitro fertilization procedures (24, 25). Increases in progesterone production occurred at physiological doses of PRL, but these effects were reversed when doses approached levels seen in hyperprolactinemic patients (22). Therefore, it is postulated that PRL might be capable of regulating genes involved in the progesterone biosynthetic pathway.
The final catalytic step in the production of progesterone is the
conversion of pregnenolone into progesterone by the enzyme,
3ß-hydroxysteroid dehydrogenase/5-4
isomerase (3ß-HSD). 3ß-HSD exists as two isoforms encoded by two
genes in humans. Type I 3ß-HSD (26) is primarily expressed in the
placenta and with lower expression in peripheral tissues such as the
prostate, breast, and skin. Type II 3ß-HSD (27, 28) is expressed in
the adrenal, ovaries, and testis. Regulation of Type II 3ß-HSD by
gonadotropins (18, 19) and PRL (29) in the rat has been demonstrated.
Human type II 3ß-HSD regulation by cAMP and phorbol esters (mimetics
of gonadotropin signaling pathways) has been reported to be dependent
upon the orphan nuclear receptor, SF-1 (30), thus providing a mechanism
for gonadotropin regulation in humans. However, direct regulation of
human type II 3ß-HSD by PRL has yet to be demonstrated. The results
herein provide a molecular mechanism by which type II 3ß-HSD
expression is regulated by PRL.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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).
This is a 9/9 match with the consensus Stat5 response element:
5'-TTCNNNGAA-3' (31).
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+46 fragment of the human type II 3ß-HSD gene linked to the
chloramphenicol acetyltransferase (CAT) reporter gene (-301CAT) was
transiently cotransfected into HeLa cells with expression vectors for
Stat5 and the PRL receptor (PRL-Rc). As shown in Fig. 2, Stat5 overexpression had little effect
on reporter activity in both untreated and PRL-treated (100 ng/ml)
cells when compared with reporter alone. PRL treatment with
PRL-Rc overexpression resulted in a 13-fold increase in
reporter activity relative to cells overexpressing PRL-Rc
without treatment. PRL treatment of cells cotransfected with Stat5 and
PRL-Rc resulted in approximately 21-fold increased reporter
activity relative to untreated cells. These results demonstrate the
ability of PRL activation of Stat5 to increase the transcriptional
activity of human type II 3ß-HSD promoter.
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, basal reporter activity
increased for -1051, -701, -301, and -101 CAT promoter constructs
but was reduced in the -52 CAT construct. PRL activation of the -1051
CAT construct yielded promoter activity that was 94-fold higher than
basal while increases in reporter activity were 23-fold and 21-fold
higher in -701 CAT and -301 CAT, respectively. PRL treatment of the
-101 CAT (P > 0.08) and -52 CAT (P
> 0.79) constructs resulted in promoter activities that were not
statistically different. The putative Stat5 response element was
deleted in the -101 and -52 CAT constructs. A similar pattern of
activation of the 5'-deletion CAT promoter constructs was seen in
experiments in which Stat5 was not overexpressed albeit at decreased
basal and PRL-stimulated levels (data not shown). These data suggest
that a major region of PRL responsiveness is eliminated with deletion
of the region containing the Stat5 response element. In addition, there
is one other region of PRL responsiveness located from -1051 -701
that does not correspond with the presence of a consensus Stat5
response element.
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-101 region containing a putative
Stat5 response element, the requirement of the Stat5 element for
transactivation of type II 3ß-HSD promoter by PRL was tested. As
shown in Fig. 1
, 3-bp point mutations were introduced into critical
base pairs in the -301 +46 CAT reporter construct converting the
element from 5'-TTCTGAGAA-3' to
5'-TTTTGATTA-3'.
Comparison of the -301(mutant) CAT promoter construct as compared with
the -301(wild-type) CAT promoter construct is shown in Fig. 4. Both reporter constructs were
transiently cotransfected into HeLa cells overexpressing Stat5 and
PRL-Rc. Basal and PRL-activated reporter activities were
dramatically reduced by mutation of the Stat5 element.
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, both basal
and PRL-treated nuclear extracts formed complex D (lane 1), which was
not further shifted by Stat5 antiserum (lane 2). This complex was
specific since it was competed away by 10x (lane 3) and 50x (lane 4)
identical, unlabeled oligonucleotide. PRL-treated extracts caused
shifting of two additional complexes B and C (lane 5). Only complex C
was supershifted (forming complex A and possibly increasing complex B)
by antiserum to Stat5 (lane 6). Both B and C were competed out by 10x
(lane 7) and 50x (lane 8) of identical, unlabeled oligonucleotide, yet
only complex C was completely competed out at 10x concentrations. Lane
9 contains an end-labeled probe that was not incubated with nuclear
extract. These experiments indicate that the Stat5 response element
forms at least one molecular complex with nuclear extracts expressing
Stat5 from cells exposed to PRL, but this complex does not form
with extracts expressing Stat5 under basal conditions.
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, all groups
were transiently transfected with 5 µg of the -301 CAT reporter
construct, 5 µg of PRL-Rc, and either increasing amounts
of Stat5 or empty expression vector yielding a total transfected
plasmid amount of 25 µg for each dish. The first group represents
PRL-induced transcriptional activity by endogenous factors. Subsequent
groups show PRL-activated promoter activity with increasing levels of
Stat5 cotransfected into the cells. Significant activation
(P < 0.001) over basal occurs with transfection at 100
ng of Stat5 and above. The overexpression of Stat5 at levels used in
other experiments (5 µg) resulted in a 5-fold increase in
PRL-activated promoter activity. These data demonstrate that increasing
the amount of Stat5 overexpressed in this system will increase type II
3ß-HSD promoter activity with a maximal response occurring above 100
ng of cotransfected Stat5 expression vector.
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, HeLa cells were cotransfected with the
-301 CAT reporter construct, and expression vectors for Stat5 and
PRL-Rc. Increasing concentrations of PRL resulted in a
dose-dependent increase in promoter activity with maximum stimulation
seen at 250 ng/ml. The response at higher doses plateau and remain
constant. These results suggest that high doses of PRL saturate the
receptors overexpressed on the cell surface, and that most likely
intracellular mechanisms do not account for inhibitory effects seen at
high doses of PRL.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-701 as seen in Fig. 3, and this increased response is
interesting in light of the absence of consensus Stat elements. Future
studies will address the potential for functional interaction between
Stat5 and heterologous transcription factors in this region and for
other mechanisms of PRL activation. Previous studies in the laboratory have identified a potential molecular mechanism by which gonadotropin regulation of 3ß-HSD occurs. Gonadotropins act through G protein-coupled receptors and activate cAMP/protein kinase A and Ca2+ flux/protein kinase C signaling pathways (34). Treatment of H295R (adrenocortical cell line) cells with phorbol esters (30) or cAMP analogs (35) will stimulate reporter gene expression when linked to the type II 3ß-HSD 5' promoter region. This activity localizes to a response element (5'-TCAAGGTAA-3') that binds the orphan nuclear receptor, SF-1, located from -64 to -56 in the 5'-flanking sequence of the transcription initiation site. Disruption of this element by inserting point mutations into critical base pairs abrogates the cAMP/phorbol ester responsiveness. It therefore appears that a major portion of gonadotropin control of 3ß-HSD occurs through the SF-1 nuclear receptor.
The relative importance of regulation of type II 3ß-HSD through the SF-1 and Stat5 response elements is unclear at this time. These sites may be working in parallel, but it is possible that formation of the transcriptional complex on the Stat5 element might interfere with formation of an SF-1 complex, thus forming the hypothesis that PRL might inhibit progesterone production via inhibition of gonadotropin signaling through the SF-1 element. Another intriguing possibility is that the Stat5 response element might be a target for other signals that activate Stat5. These include growth factors (i.e. GH or EGF) or cytokines. These factors might up-regulate type II 3ß-HSD enzyme levels under physiological or pathological conditions. Stat5 has also been shown to interact with nuclear receptors. For instance, the glucocorticoid receptor has been shown to functionally interact with Stat5 in up-regulation of ß-casein expression in the presence of the lactogenic hormones: insulin, hydrocortisone, and PRL (36). Identification of the Stat5 response element in the type II 3ß-HSD promoter opens the possibility that transduction of other signals might occur through this element either by directly activating Stat5 or interacting with Stat5 via protein-protein interactions.
Regulation of the type II 3ß-HSD gene by PRL provides a
mechanism by which PRL can elicit some of its ovarian effects. Factors
that regulate luteal function are either luteotropins or luteolysins
that increase or decrease progesterone output by the CL, respectively.
The gonadotropins LH (rat) and LH/hCG (human) have been known to play a
luteotrophic role by acting through G protein-coupled transmembrane
receptors and elevating protein kinase A and protein kinase C
activities (34). PRL has been shown to have a dual role in luteal
function in the rat. Depending upon experimental conditions, PRL
treatment of hypophysectomized rats has been shown to induce either
luteolysis (19, 29) or be luteotrophic (37). Further evidence for
a luteotrophic role of PRL in the rat ovary comes from studies in
which 20-hydroxysteroid dehydrogenase (20-HSD), an enzyme that
metabolizes progesterone into an inactive metabolite, was shown to be
down-regulated by PRL (17). These studies support the idea that PRL
plays a role in both the maintenance and degradation of the rodent CL
depending upon experimental conditions used.
The role PRL plays in the primate ovary is equally complex. Studies of the effect of PRL exposure to granulosa-lutein cell cultures demonstrated that PRL is required at low doses (<20 ng/ml) for progesterone production by these cells, yet progesterone production is inhibited at higher PRL concentrations (>20 ng/ml) (22, 24, 25). This reduction in progesterone production occurs with PRL levels that correlate with concentrations seen in women with hyperprolactinemia (8). Other studies have shown that reduction of PRL by bromocriptine treatment resulted in shorter luteal cycles and reduced serum progesterone levels in women (7). These studies suggest an analogous dual-function role of PRL in either maintenance or disruption of progesterone output by the human CL, and that PRL effects upon the CL differ depending on circulating concentrations of the hormone. Regulation of human type II 3ß-HSD by PRL could account for the reduction in progesterone production by luteal cells and reduced serum progesterone levels in hypoprolactinemic women.
The importance of the PRL-Rc/Stat5 signaling system in
mediating the effects of PRL in the female reproductive system has been
demonstrated in gene knockout experiments. Stat5 proteins are expressed
in two isoforms that arise from two separate genes: Stat5a and Stat5b
(38, 39). Mice deficient in Stat5a do not lactate and are fertile (40).
Mice deficient in Stat5b show impaired mammary gland development and
spontaneous abortions that can be rescued by the administration of
progesterone (41). Apparent compensation of some Stat5 function occurs
in either knockout because Stat5a/Stat5b double knockout mice have a
more severe reproductive phenotype. These mice are infertile, do not
form corpora lutea, and show an up-regulation of 20-HSD (42). Stat5
levels have also been shown to increase in the ovaries of
pseudopregnant rats (32). Mice lacking PRL-Rc are sterile,
show irregular cycles, and do not exhibit pseudopregnancy (6), and mice
lacking the PRL gene are infertile (5). These experiments clearly
demonstrate the importance of Stat5, PRL, and PRL-Rc in
regulating luteal function.
In summary, these data demonstrate the stimulatory effect of PRL on human type II 3ß-HSD promoter activity. PRL induces promoter activity by the formation of a transcriptionally active complex on the Stat5 response element in the promoter that contains Stat5. This activity is disrupted by the introduction of specific point mutations into the Stat5 element or by its deletion. These data provide a potential mechanism by which human type II 3ß-HSD can be up-regulated in response to PRL treatment, accounting for increases in progesterone production by human luteal cells by PRL in culture and reduced progesterone output by women with decreased circulating PRL due to bromocriptine treatment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transient Transfection
HeLa cells were transiently transfected using a modification of
the calcium phosphate coprecipitation method (43). Plasmid constructs
employed were ovine Stat5 and the murine PRL receptor (long form)
subcloned into pcDNA3 (Invitrogen, San Diego, CA)
expression vectors. Human type II 3ß-HSD promoter fragments were
inserted into pCAT-Basic (Promega Corp., Madison, WI)
reporter plasmids as described previously (30). Adherent HeLa cells
were cultured to 55–65% confluency in 100-mm tissue culture dishes
(Corning, Inc., Corning, NY) in 10 ml of the appropriate medium.
Calcium phosphate-DNA coprecipitates were formed by dropwise addition
of equal volumes (0.5 ml) of solution A (0.24 M
CaCl2 containing 15 µg of appropriate plasmid constructs)
to solution B [2x HEPES-buffered saline; 50 mM HEPES, 1.4
mM Na2HPO4, 0.28 M NaCl
(pH 7.1)]. Calcium phosphate-DNA precipitates were incubated at 23 C
for at least 20 min and added to single 100-mm dishes of cells
containing 9 ml of fresh medium. HeLa cells were then incubated with
precipitate for 4 h at 37 C (5% CO2 and 95% air),
shocked for 1 min with 15% (vol/vol) glycerol in Dulbecco’s PBS
(D-PBS; 0.137 M NaCl, 0.5 mM
MgCl2, 6.45 mM Na2HPO4,
1.5 mM K2HPO4), washed three times
with D-PBS, and incubated at 37 C for 24 h. During the final
24 h of incubation, cells were cultured in the presence or absence
of appropriate treatment. Cells were then harvested using trypsin/EDTA
(Life Technologies), pelleted, resuspended in 0.25
M Tris-HCl (pH 7.4), and stored at -70 C until assayed for
CAT activity. Transfections were performed in triplicate with mock
negative controls. Internal transfection efficiency was monitored by
cotransfection of 1 µg of either pSEAP2 (secreted alkaline
phosphatase) or pCMV-ß-galactosidase constructs and measurement of
respective enzymatic activities. One hundred percent transfection
efficiency was considered to be the group with the highest control
enzymatic activity (alkaline phosphatase or ß-galactosidase), and all
groups were normalized to this value.
CAT Assays
Frozen cell pellets were thawed on ice and lysed by sonication.
Extracts were heated to 60 C for 5 min to denature any endogenous
acetylase/deacetylase enzymes. Soluble extracts were then separated
from cell debris by centrifugation, divided into aliquots for CAT
assays, and stored at -70 C before use. Fluorescent CAT assays were
performed as described previously (44) with some modifications using
the FLASH CAT assay kit (Stratagene, La Jolla, CA). Acetyl
coenzyme A (CoA) was synthesized by reaction of CoA (Pharmacia Biotech, Piscataway, NJ) with acetic anhydride (Sigma Chemical Co.) as described elsewhere (45) and stored at -70 C
until use. Cell extracts (10–20 µl) were incubated in 0.25
M Tris-HCl (pH 7.4) in a total reaction volume of 50 µl
with acetyl-CoA (8.2 µM) and fluorescent
borondipyrromethene difluoride (BODIPY) chloramphenicol (CAM) substrate
(1:12.5 dilution) at 37 C for 4–8 h. Reactions were terminated by
addition of cold ethyl acetate (850 µl) followed by vigorous
vortexing. An aliquot (800 µl) of extracted substrate and acetylated
products was removed (organic phase), dried under vacuum, and
resuspended in ethyl acetate (20 µl) before separation on TLC plates
(LK6, Whatman, Clifton, NJ) with chloroform-methanol (9:1)
for 30 min. Substrate and products were visualized under long-wave UV
light (366 nm) and photographed (type 55 positive/negative film,
Polaroid). Substrate and combined product bands were scraped from the
plates, extracted, and diluted 1:10 in methanol before quantification
by fluorescence spectrophotometry at excitation and emission
wavelengths of 490 nm and 512 nm, respectively, using a fluorometer.
Percent conversion of BODIPY CAM substrate to 1-, 3-, and
1,3-acetylated BODIPY CAM products was computed.
Preparation of Nuclear Extracts
Crude nuclear extracts were prepared as described previously
(46) with modifications (47). Three identical 100-mm tissue culture
dishes of HeLa cells were transfected and cultured as described. At
55–65% confluency, cells were cultured in the presence or absence of
100 ng/ml ovine PRL for 20 min and harvested by scraping into D-PBS
containing tyrosine phosphatase inhibitors (1 mM
Na3VO4, 50 µM
Na3MoO4). Cells were then pelleted and
resuspended in Buffer A [10 mM HEPES-KOH (pH 7.9), 1.5
mM MgCl2, 10 mM KCl, 0.5
mM dithiothreitol, 0.2 mM phenylmethylsulfonyl
fluoride, 1 mM Na3VO4, 50
µM Na3MoO4] for 10 min at 4 C
followed by vortexing and centrifugation. The supernatant was then
discarded and the pellet resuspended in Buffer C (20 mM
HEPES-KOH (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5
mM MgCl2, 0.2 mM EDTA, 0.5
mM dithiothreitol, 0.2 mM phenylmethylsulfonyl
fluoride, 1 mM Na3VO4, 50
µM Na3MoO4) for 20 min to extract
nuclear proteins. The suspension was centrifuged, and the supernatant
containing nuclear proteins was aliquoted and stored in liquid
nitrogen. Protein concentrations were determined using the BCA method
(Pierce Chemical Co., Rockford, IL).
Electrophoretic Mobility Shift Analysis (EMSA)
EMSA experiments were performed as described (48) with some
modification. Single-stranded, complementary 30-bp oligonucleotides
(5'-GTCACTATTATTCTGAGAAAAGGGATTCTG-3')
containing the putative Stat5 response elements were synthesized
(Life Technologies, Inc.). Double-stranded probes were
prepared by annealing 50 ng of each oligonucleotide strand for 2 min at
95 C, followed by slow cooling to room temperature. The probe was then
end-labeled using [
-32P]ATP (3000 Ci/µmol;
Amersham, Arlington Heights, IL) and T4 polynucleotide
kinase (New England BioLabs, Inc., Beverly, MA) and
purified using Nuc-Trap columns (Stratagene). Nuclear
extracts (20 µg) from cells were preincubated in the presence (1
µl) or absence of anti-Stat5 (C-17; Santa-Cruz Biotechnology, Inc.,) for 30 min on ice before the addition of
poly(dIdC)·poly(dIdC) (2 µg, Pharmacia Biotech) in
15.0 mM HEPES (pH 7.9), 50 mM KCl, 42
mM NaCl, 0.15 mM MgCl2, 1
mM EDTA, 1 mM dithiothreitol, 2.5% glycerol,
4% Ficoll, 32P-labeled oligonucleotide (
4 x
104 cpm) to a final reaction volume of 20 µl and
incubated for an additional 30 min on ice. In additional competition
experiments, reactions contained unlabeled, double-stranded
oligonucleotide (10x or 50x molar excess). DNA-protein complexes were
resolved using native PAGE (5% acrylamide-bisacrylamide, 37.5:1) with
0.5x Tris borate-EDTA (44.5 mM Tris, 44.5 mM
boric acid, 1 mM EDTA) for 2 h at 150 V. Gels were
then dried under vacuum at 70 C for 1 h and exposed to Kodak
BioMax MR film (Eastman Kodak Co., Rochester, NY) for
24 h at -70 C.
Generation of Mutated Stat5 Response Element
The -301 (mutant) CAT promoter construct was generated by
synthesizing (Life Technologies) overlapping top and
complementary DNA strands with directional restriction endonuclease
sites at either end. The oligonucleotides, ranging in size from 19 to
45 bases, modified the Stat5 element from 5'-TTCTGAGAA-3' to
5'-TTTTGATTA-3'. The oligonucleotides were
phosphorylated by T4 polynucleotide kinase, ligated with
T4 DNA ligase, and then filled with the Klenow fragment of
DNA polymerase. The final blunt fragment was cleaved with restriction
endonucleases, agarose gel-purified, and ligated into pCAT-basic. The
final -301 (mutant) CAT promoter construct was sequenced on both
strands to verify the point mutations and the fidelity of the remaining
sequence.
Statistical Analysis
Statistical significance was determined by single-factor ANOVA
followed by Bonferroni correction for multiple comparisons. Sample
differences were not considered to be statistically significant unless
P < 0.05 (divided by the number of treatment groups)
as per the Bonferroni correction for multiple comparisons.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Frank A. Feltus was supported in part by NIH Training Grant 5T3-HD-07043.
Received for publication February 1, 1999. Revision received March 19, 1999. Accepted for publication March 31, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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