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Laboratory of Molecular Endocrinology (O.B., E.L., D.W.H.), Medical Research Council Group in Molecular Endocrinology (A.B.), Centre Hospitalier de L’Université Laval Research Center, Laval University, Québec, Canada, G1V 4G2
Address all correspondence and requests for reprints to: Dr. Dean W. Hum or Dr. Alain Bélanger, Laboratory of Molecular Endocrinology, Centre Hospitalier de L’Université Laval Research Center, 2705 Laurier Boulevard, Québec G1V 4G2, Canada. E-mail: dean.hum@crchul.ulaval.ca; or alain.belanger{at}crchul.ulaval.ca
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Abstract |
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-Diol, estriol, and 4-hydroxyestrone, respectively. RT-PCR
analysis revealed that UGT2B23 transcript is expressed in several
tissues, including the prostate, mammary gland, epididymis, testis, and
ovary. Primary structure analysis shows that UGT2B23 is in the same
family of enzymes as the previously characterized monkey isoforms
UGT2B9 and UGT2B18, which are active on hydroxyandrogens. The
characterization of UGT2B23 as a functional enzyme active on
3-hydroxysteroids, and its expression in extrahepatic tissues,
indicate that it may potentially play an important role in estrogen and
androgen catabolism in peripheral steroid target tissues. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In lower mammals, the testis is the exclusive source of androgens;
whereas in humans, the adrenals secrete large amounts of the androgen
precursors dehydroepiandrosterone (DHEA) and
DHEA-sulfate. These precursor steroids are converted into
potent androgens and estrogens in peripheral tissues (such as the
prostate, breast, and skin) by steroidogenic enzymes, which include
3ß-hydroxysteroid dehydrogenase, 17ß-hydroxysteroid dehydrogenase,
and 5-reductase (5, 6). Moreover, it is now clear that extrahepatic
tissues in humans also express steroid-conjugating UGT enzymes (1, 2, 7, 8). Based on the expression of UGT2B transcripts in most (if not
all) steroid target tissues (9), and the significant concentration of
glucuronidated steroids found in these tissues and in the circulation
of humans, it has been proposed that UGT enzymes can glucuronidate
steroids and contribute to modulate the steroid response in
extrahepatic steroid target tissues (7, 9).
In humans, significant levels of steroid metabolites, in the form of
glucuronide conjugates, are detected in circulation. It has been
suggested that the plasma levels of 5-reduced C19
steroid glucuronides, androsterone-glucuronide (ADT-G), and
5-androstane-3,17ß-Diol-G (3-Diol-G) reflect the peripheral
tissue conversion of adrenal and gonadal precursor C19
steroids to active androgens (1). A recent study suggests that the
serum levels of androgens or estrogens during aging are a poor
indicator of total androgenic and estrogenic activities in men and
women (10); whereas, the level of circulating conjugated androgen
metabolites was shown to be correlated with the total androgen pool in
men (10). In addition, the plasma levels of steroid glucuronides are
increased in some hyperandrogenic pathologies, such as acne or
hirsutism, which are related to the increased production of
5-reduced C19 steroids (11). In women with hirsutism,
the increased plasma concentration of 5-reduced C19
steroid glucuronides may be a reflection of increased C19
steroid metabolism in peripheral tissues.
Based on the homology of primary structures, the mammalian UGT proteins have been categorized into two major families, UGT1 and UGT2; with the UGT2 family further divided into two groups, UGT2A and UGT2B (3). Enzymes of the UGT2B subfamily catalyze the glucuronidation of several endogenous compounds, including bile acids, steroids, fatty acids, and carboxylic acids (3). The characterizations of human UGT2B enzymes demonstrate an overlap of substrate specificities between the different proteins. However, each enzyme is active on specific classes of steroids, which are glucuronidated at their hydroxyl groups.
As found in humans, the monkey also has high plasma levels of
androgen-glucuronides (12), which indicates that simians represent a
relevant animal model for studying steroid glucuronidation. The
isolation and characterization of simian UGT2B enzymes are important
steps required to gain an understanding of the role of glucuronidation
in steroid metabolism. In the present study, we report the isolation
and characterization of a novel UGT enzyme, UGT2B23, which is expressed
in several steroid target tissues, and which glucuronidates androgen
(ADT, 3-Diol) and estrogen metabolites (estriol,
4-hydroxyestrone).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Diol) were purchased from NEN Life Science Products (Boston, MA). Radioinert steroids were purchased from
Steraloids Inc. (Wilton, NH). [14C]UDPGA (285 mCi/mmol)
was obtained from NEN Life Science Products, and
-[32P]-dCTP (3000 Ci/mmol) was from
Amersham Pharmacia Biotech (Ontario, Canada). Geneticin
(G418) and Lipofectin were obtained from Life Technologies
(Ontario, Canada). Protein assay reagents were obtained from
Bio-Rad Laboratories, Inc. (Richmond, CA). Restriction
enzymes and other molecular biology reagents were from Pharmacia LKB Biotechnology Inc. (Milwaukee, WI), Life Technologies, Stratagene (La Jolla, CA) and
Roche Molecular Biochemicals (Indianapolis, IN). AmpliTaq
DNA polymerase was from Perkin-Elmer Cetus (Branchburg,
NJ) and Roche Molecular Biochemicals. Human embryonic
kidney 293 cells (HK293) were obtained from the American Type Culture Collection (Rockville, MD).
Monkey RNA isolation
Total RNA was isolated from monkey mammary gland, epididymis,
prostate, kidney, testis, adrenal, bile duct, small intestine, brain,
cerebellum, lung, colon, spleen, liver, ovary, seminal vesicle,
thyroid, vagina, HK293 cells, and HK293 cells stably expressing
UGT2B23, according to the Tri reagent acid phenol protocol as specified
by the supplier (Molecular Research Center, Inc.,
Cincinnati, OH). The messenger RNAs (mRNAs) obtained from a monkey
liver tissue was obtained by affinity chromatography through oligo
(dT)-cellulose (Pharmacia LKB Biotechnology Inc.).
Complementary DNA (cDNA) isolation
Affinity-purified liver mRNA was used to construct a cDNA
library in the 
ZAP express vector, as specified by the
manufacturer’s instructions (Stratagene). The library was
not amplified for screening as previously described (8). The UGT2B23
cDNA clone was excised from the pBK-CMV vector using an helper phage
(Stratagene). UGT2B23 cDNA clone was isolated and was
sequenced in both directions, using specific UGT oligonucleotides
(8).
Transcription/translation, in vitro, of the UGT2B23 cDNA
The entire UGT2B23 cDNA in the pBK-CMV vector was transcribed
and translated using T3 RNA polymerase in the
transcription/translation-coupled rabbit reticulocyte lysate system
from Promega Corp. (Madison, WI) in the presence of
[35S]-methionine. The protein product was separated on a
12% SDS/polyacrylamide gel and exposed on Hyperfilm-MP for 1
h.
Stable expression of UGT2B23
HK293 cells were grown in DMEM containing 4.5 g/l glucose, 10
mM HEPES, 110 µg/ml sodium pyruvate, 100 IU
penicillin/ml, 100 µg/ml streptomycin, and 10% FBS in an humidified
incubator, with an atmosphere of 5% CO2, at 37 C. Five
micrograms of pBK-CMV-UGT2B23 was used to transfect HK293 cells using
Lipofectin. A stable transfectant was selected in media containing 1
mg/ml G418, as previously reported (13).
Microsomal proteins isolation
Next, 8 x 106 transfected HK293 cells were
homogenized in 5 ml homogenization buffer (14) and centrifuged at
12,000 x g, at 4 C, for 20 min. The supernatant was
centrifuged at 105,000 x g for 1 h at 4 C. The
microsome pellets was resuspended in 0.5 ml of homogenization
buffer.
Endoglycosidase H digestion
Microsomal proteins from monkey liver (5 µg), ovary (25 µg),
HK293 cells (25 µg), and HK293 cells stably expressing UGT2B23 (25
µg) were incubated with 20 mU endoglycosidase H, in the presence of
50 mM sodium acetate (pH 5.5) and 0.1% SDS, in a final vol
of 20 µl for 16 h at 37 C. Proteins were separated on a 12%
SDS-polyacrylamide gel, transferred onto nitrocellulose membrane, and
probed with the EL-93 anti-UGT2B17 antisera (1:1500 dilution), as
reported (15). An antirabbit IgG horse antibody conjugated with
peroxidase (Amersham Pharmacia Biotech was used as the
second antibody, and the resulting immunocomplexes were visualized
using a chemiluminescence kit (ECL) (Renaissance, Québec, Canada)
and exposed on hyperfilm for 1 min (Eastman Kodak Co.,
Rochester, NY).
Glucuronidation assay and Michaelis constant
(Km) determination using cell homogenates and
microsomal proteins
HK293 cells expressing exogenous UGT2B23 were suspended in Tris
buffered saline containing 0.5 mM dithiothreitol and were
homogenized using a Brinkmann Instruments, Inc. (Westbury,
NY) polytron. For a first assessment of activity, enzyme assays
were performed using 7.5 µM [14C]UDPGA,
92,5 µM unlabeled UDPGA, 200 µM of the
various aglycons, and 150 µg protein from cell homogenates in 50
mM Tris-HCl (pH 7.5), 10 mM MgCl2,
100 µg/ml phosphatidylcholine, and 8.5 mM saccharolactone
in a final vol of 100 µl. Assays were performed for 16 h at 37 C
and were terminated by adding 100 µl methanol. Chromatography
analysis and formation of glucuronide were determined as previously
described (8).
Compounds that demonstrated reactivity with UGT2B23 in the screening assay were subsequently reassayed with HK293-UGT2B23 microsomal proteins in 50 mM Tris-Hcl (pH 8), for 4 h at 37 C, in the presence of 7.5 µM [14C]UDPGA, 492.5 µM unlabeled UDPGA. Under these conditions, the enzyme reaction is linear for 6 h. The values represent the means of two independent experiments performed in triplicates. Kinetics analysis was realized, using microsomal proteins in the same conditions, except that final concentrations of estriol, 4-hydroxyestrone, and etiocholanolone were 1, 2, 4, 6, 8, 10, and 15 µM.
Km determination in intact HK293 cells
Km determinations were performed by incubating
intact HK293 cells with radiolabeled steroid substrates, as previously
described (13). Cells were incubated for 6 h at 37 C with 0.25,
0.5, 0.75, 1, 2.5, and 5 µM radioinert ADT or 3-Diol
and 20 nM of corresponding radiolabeled substrate. The
medium was removed and analyzed for glucuronide conjugates by organic
extraction and scintillation counting, as previously described (16).
The data obtained were normalized by DNA content quantitated by
fluorometric assay with 3,5-diaminobenzoic acid (17).
RT-PCR analysis
The tissue distribution of UGT2B23 was achieved
using a RT-PCR technique, as previously reported (18). Five micrograms
of total RNA from cynomolgus monkey tissues HK293 cells and HK293 cells
stably expressing UGT2B23 were used. Reverse transcriptase reactions
were performed using 500 pmol of the UGT2B23-specific antisense primer
5'-GAAAAGAAATCCTCCACAATGCTTTTCAAAAACA3' and 2 pmol of the control
antisense GAPDH primer, 5'-CCCAGCGTCAAAGGTGG-3', in the presence of 200
U SuperScript II reverse transcriptase, according to the
manufacturer’s instructions (Life Technologies). The PCR
reaction was carried out with one fourth of the complementary RNA
product and 100 pmol of the specific sense primer
5'-CCTGAGTTTGAGAATATAGTCACGCAAGAGA3' using ampli Taq DNA
polymerase (Perkin-Elmer Cetus). The PCR was performed for
30 cycles (1 min at 94 C, 1 min at 67 C, 1 min at 72 C), after which
one fifth of the PCR product was electrophoresed on an ethidium
bromide-stained 1% agarose gel. The sense and antisense primers start
at positions 250 and 567, respectively, in relation to the adenine of
the initiator codon, which is designated nucleotide 1 (Fig. 1
). All PCR reactions were controlled
using the sense primer for GAPDH, 5'-TGGGTGTGAACCATGAG-3'. The identity
of all PCR products was verified by direct sequencing (19).
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Results |
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). Two putative polyadenylation
signals are present at nucleotides 1724 and 1735, with a
poly(A+) tail starting at position 1750. The nucleotides
proximal to the first AUG codon is consistent with it being the
initiator methionine, according to the findings of Kozak (20). The
protein primary structure of 529 amino acids, which is deduced from the
nucleotide sequence, reveals the presence of several conserved
sequences found in most of the UGT2B enzymes (Fig. 1) (3). The UGT2B23
protein contains a characteristic hydrophobic signal peptide from
residue 1–23, a hydrophobic transmembrane region between amino acids
494 and 510, and 2 potential asparagine glycosylation sites
[NX(S/T)] present at positions 68 and 69 (Fig. 1).
Amino acid sequence alignments show that UGT2B23 is 94%
identical to the previously characterized monkey UGT2B9 and UGT2B18
isoenzymes (Fig. 2). Of the 32 amino acid
differences found between UGT2B23 and UGT2B18, 10 are found in the
carboxylterminal domain between residues 291 and 530, which was
proposed to bind the cofactor UDPGA (Fig. 1). Comparisons with
UGT2B9 reveal 13 different residues in the carboxylterminal domain,
whereas 18 variant residues are found in the aminoterminal half of the
proteins. UGT2B23 is 82, 87, 77, and 76% identical to the human
UGT2B4, UGT2B7, UGT2B15, and UGT2B17 isoenzymes, respectively (Fig. 2).
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). To
characterize the UGT2B23 protein, the cDNA was stably transfected into
HK293 cells, and the derived cell line was demonstrated to express a
51-kDa protein, by Western blot analysis of microsomal proteins (Fig. 3B). Microsomes from monkey liver and ovary also contain proteins of
similar apparent molecular mass, which are recognized by the
anti-UGT2B17 antibody. In contrast, untranfected control HK293 cells
did not express immunoreactive proteins. To determine whether the
UGT2B23 protein was glycosylated, microsomal protein from HK293 cells
stably expressing UGT2B23 was treated with endoglycosidase H.
Immunoreactive protein from the liver and ovary displayed faster
migration after endoglycosidase H treatment, indicative of glycosylated
UGT2B enzyme. However, the migration of UGT2B23 remained the same,
which suggests that this protein is not glycosylated (Fig. 3B). When
separated on the same gel by SDS-PAGE, the proteins UGT2B9, UGT2B18,
and UGT2B23 all migrated at the same rate but faster than the UGT2B20
protein (data not shown), which was previously found to be glycosylated
(13). Considering that all four proteins are comprised of 528–530
residues, this result is consistent with the possibility that UGT2B9
and UGT2B18, which have two potential overlapping glycosylation
sites identical to UGT2B23, are not glycosylated; whereas UGT2B20,
which has four different potential glycosylation sites, is
glycosylated.
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). Of the 62 different endogenous
compounds tested, only 7 were conjugated by UGT2B23 (Table 1). Glucuronidation of these compounds
was not detected using control HK293 cell homogenates. The main
substrates of UGT2B23 are 3-hydroxyandrogens (etiocholanolone and
3-Diol), 4-hydroxyestrone, estriol, and the bile acid HDCA
(hyodeoxycholic acid); however, UGT2B23 does not glucuronidate
neuromodulators and C21 steroids. As found with most other
UGT2B enzymes, UGT2B23 is active on planar phenols, including
4-propyl-phenol, 1-naphthol, and eugenol (data not shown).
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-Diol, and ADT,
was performed using intact HK293 cells stably expressing UGT2B23 (Fig. 5B). The apparent Km value for
conjugation of 3-Diol (13.5 µM) is more than 10-fold
higher than for ADT (0.9 µM), as determined by double
reciprocal plots (Lineweaver-Burk). The apparent maximal
velocity (Vmax) values obtained for these two substrates
were similar, thus the Vmax/Km ratio demonstrates a 10-fold
higher efficiency for the glucuronidation of ADT. Similar experiments
performed using microsomal proteins demonstrate Km values of 3.5, 1.6,
and 5.7 µM for etiocholanolone, estriol, and
4-hydroxyestrone, respectively (Fig. 5A). The Vmax/Km ratio
is similar for estriol and etiocholanolone, indicating the same
efficiency of glucuronidation, whereas 4-hydroxyestrone presents a
5-fold lower efficiency of conjugation by UGT2B23.
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).
Identity of the 318-bp UGT2B23 RT-PCR product was confirmed by direct
sequencing. The integrity of each RNA sample was verified by
amplification of the GAPDH transcript, using specific
oligonucleotides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reduced C19 steroid glucuronides among nine mammalian
species, it was found that humans and simians are unique in having
significant levels of ADT-G and 3-Diol-G (12). Moreover, several of
the extrahepatic tissues in these two species were shown to express
steroid-conjugating UGT2B transcripts (14). In addition, the cloning
and characterization of other steroidogenic enzymes (such as
5-reductase from the rhesus monkey) demonstrated high identity in
primary structure and very similar biochemical properties as the human
protein (21). Thus, taken together, these data suggest that the monkey
is an appropriate animal model in which to study steroidogenesis and
the role of steroid glucuronidation in extrahepatic steroid target
tissues. Similar to several of the human UGT2B enzymes, the ability of
UGT2B23 to conjugate androgens and estrogens is consistent with its
potential role in steroid metabolism.
Of the 13 different androgens tested as potential substrates in
this study, glucuronidation by UGT2B23 was detected only on
C19 steroids, which are hydroxylated at the 3-position
(etiocholanolone, ADT, and 3-Diol) (Table 1 and Fig. 4).
Interestingly, the glucuronidation of 3ß-hydroxylated androgens was
not detected, which suggests a differential specificity of UGT2B23 for
different stereoisomers of the C19 steroid molecule.
UGT2B23 conjugates 5- and 5ß-reduced androgens and is in contrast
to the monkey UGT2B19 enzyme (18), which is more active on 5ß-reduced
compounds (Table 2).
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), including UGT2B9 (94% identity)
and UGT2B18 (94% identity). In contrast to UGT2B23, which is specific
for 3-hydroxylated steroids, UGT2B9 is capable of glucuronidating
steroids at both the 3-hydroxy and 17ß-hydroxy positions [ADT,
testosterone, and dihydrotestosterone (DHT)] (14), whereas UGT2B18 is
specific for 3-hydroxyandrogens and does not conjugate
C18 steroids (22) (Table 2). These differences in substrate
specificity are most likely conferred by differences in primary
structure at the aminoterminal halves of the proteins, which have been
proposed to contain the aglycon binding site. More specifically, the
region between residues 90 to 229 contains variant amino acids at the
identical position of the UGT2B9, UGT2B18, and UGT2B23 proteins (Fig. 1).
In humans, UGT2B4 (23, 24), UGT2B7 (23), UGT2B15 (25), and UGT2B17 (8)
are steroid-conjugating enzymes. However, despite the high homology
between UGT2B proteins from the two species, it is interesting that
there exist differences in their steroid specificity. For example,
UGT2B7, which is the human protein most homologous to UGT2B23, is
active on 3-hydroxyandrogens, estrogen metabolites (estriol,
2-hydroxyestrone, 4-hydroxyestrone), and C21 steroids (23).
It is clear that the steroid specificity of UGT2B enzymes between two
species, or even within the same species, cannot be predicted based on
sequence homology. In addition, the rat UGT2B2 enzyme has the same
androgen specificity as the monkey UGT2B23, in that they can both
conjugate ADT (3-OH) and not DHT nor testosterone (17ß-OH) (26).
Thus, based on steroid substrate specificity, it is possible that a
human orthologue of UGT2B23 remains to be isolated and
characterized.
Androgens have important physiological functions, including the
regulation of sex organ development in the embryonic stage and
acquisition of male secondary sexual characteristics at puberty. In
adult life, androgens are involved in maintaining homeostasis; however,
in certain pathologies (such as prostate cancer) C19
steroids can have deleterious effects and lead to increased cancer cell
proliferation. In peripheral steroid target tissues [such as the
breast, skin and prostate, which express UGT2B transcripts (8, 25)],
it is clear that the level of steroids is regulated by anabolic
and catabolic enzymes. In these tissues, it has been proposed that
glucuronidation of steroids abolishes their specific interaction with
nuclear receptor and leads to their excretion from the tissue (1). As
determined by RT-PCR, UGT2B23 transcript is expressed in steroid target
tissues, which is consistent with this enzyme being involved in steroid
catabolism at these sites. UGT2B23 transcript was not detected in the
seminal vesicle, which is a known androgen-responsive tissue. However,
considering that the family of UGT2B enzymes has overlapping (but
distinct) patterns of substrate specificity, it is possible that other
androgen-conjugating UGT2B proteins are expressed in this tissue.
UGT2B23 can glucuronidate ADT with a 10-fold higher efficiency (ratio
Vmax/Km) than 3-Diol, which correlates with the 10:1
ratio of ADT-G/3-Diol-G found in the circulation (12). This 10:1
ratio is also observed in human plasma, but the human
steroid-conjugating UGT2B enzymes characterized to date do not exhibit
a 10-fold higher efficiency of conjugation for ADT over 3-Diol.
Similar to androgens, estrogens exert many important functions via interaction with their receptors (27). The catabolism of estrogens include oxidative metabolism and conjugative metabolism, by glucuronidation, sulfonation, and O-methylation, to decrease the effect of the parent hormone (28). The activity of UGT2B23 on estriol, with an apparent Km value of 1.6 µM, and its expression in extrahepatic estrogen-sensitive tissues (such as the breast, ovary, and prostate) is consistent with this enzyme playing a role in steroid metabolism. Conjugation of estrogens by UGT2B23 in these tissues may be required to promote steroid elimination to maintain homeostasis or to increase elimination required after increased estrogen synthesis in response to physiological conditions.
The high activity of UGT2B23 on 4-hydroxyestrone, with an apparent Km value in the micromolar range, suggests a physiological role of this enzyme on the catecholestrogen. Although 2-hydroxylation of estradiol and estrone is the dominant pathway for catecholestrogen formation in liver microsomes, recent data indicate that formation of 4-hydroxylated catecholestrogens is a dominant pathway in several extrahepatic steroid target tissue, such as the breast and uterus (29, 30). Glucuronidation of the catecholestrogen 4-hydroxyestrone is potentially an important catabolic pathway required to eliminate these genotoxic steroid metabolites from a given tissue and to prevent cell damage (31, 32, 33). Catecholestrogens can undergo metabolic redox cycling catalyzed by P450 enzymes. The hydroperoxide-dependent oxidation of catecholestrogens to quinones, and the NADPH-dependent reduction of the quinones back to hydroquinones, yield semiquinone free radical intermediates and superoxide radicals (34). The continuous generation of the free radicals by the redox cycle have been postulated to mediate DNA damage (such as single-strand breaks, 8-hydroxylation of guanine bases, and depurination of adenine-guanine adducts) leading to tumor development (31, 34, 35). This potential problem of catecholestrogens is particularly relevant in estrogen sensitive-tissues (such as the breast, ovary, and uterus) that express steroidogenic enzymes (including aromatase required for estrogen synthesis) and the enzymes (such as cytochrome P4501B1) that yield catecholestrogens (30, 36, 37, 38). It is interesting that several steroid-specific UGT enzymes (including UGT2B23) are also expressed in these tissues; thus, it will be important to determine the role that each of these proteins may have in metabolizing estrogens and, subsequently, influencing their effects.
In summary, the present study demonstrates that the simian UGT2B23
enzyme is capable of conjugating specific 3-hydroxylated
C18 and C19 steroids. The glucuronidation of
steroids by UGT2B23 with apparent Km values in the low micromolar range
and the expression of its transcript in the breast, ovary, testis, and
prostate suggest that this enzyme plays a role in steroid metabolism in
extrahepatic steroid target tissues. The high homology between the
simian and human proteins, and the ability of these enzymes to
conjugate steroids, correlates well with the finding that these two
species have similar mechanisms of steroid glucuronidation and are thus
far unique in containing significant amounts of glucuronidated androgen
metabolites in the plasma. Characterization of the simian UGT2B enzymes
establishes the monkey as a relevant animal model for further study, to
understand the role and significance of steroid glucuronidation in
steroid target tissues.
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Acknowledgments |
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Received April 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reduced steroid glucuronides in
hyperandrogenic and idiopathic hirsute women. Fertil Steril 48:948–953[Medline]
-reduced C19 steroid glucuronides in
primates, rodents, and domestic animals. Am J Physiol
271:E348–E353
-reductase: evidence for mechanism-based inhibition
by finasteride. J Steroid Biochem Mol Biol 66:271–279[CrossRef][Medline]
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