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-Like Protein in Pancreatic Islets: Molecular Cloning and Expression Pattern during Development and Growth of the Endocrine Pancreas1
Hagedorn Research Institute, Gentofte (C.C., K.L., P.G., N.B., B.M., J.H.N.), and the Department of Molecular Cell Biology, Statens Seruminstitut (D.I., L.-I.L.), Copenhagen, Denmark
Address all correspondence and requests for reprints to: Dr. Carina Carlsson (nee Svensson), Department of Medical Cell Biology, Box 571, Biomedical Center, Uppsala University, 751 23 Uppsala, Sweden. E-mail Carina.Carlsson{at}medcellbiol.uu.se
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-like protein (Dlk)]. The
size of Pref-1 messenger RNA (mRNA) in islets was 1.6 kilobases, with
two less abundant mRNAs of 3.7 and 6.2 kilobases. The Pref-1 mRNA
content of islets from adult rats was only 1% of that in neonatal
islets. Pref-1 mRNA was markedly up-regulated in islets from pregnant
rats from day 12 to term compared with those from age-matched female
rats. Two peaks in mRNA expression were observed during gestation, one
on day 14 and the other at term, whereafter it decreased to nonpregnant
levels. Pref-1 mRNA was up-regulated 3- to 4-fold in neonatal rat
islets of Langerhans after 48-h culture with hGH, as found also with
bovine GH or ovine PRL. During the development of pancreas from
embryonic day 12 (E12) to postnatal day 4, we observed a 2-fold
increase in Pref-1 mRNA on E17 and a 5-fold increase at birth, followed
by a rapid decline on postnatal day 4. Pref-1 immunoreactivity was
found in a subpopulation of insulin cells of neonatal islets of
Langerhans. At an early embryonal stage (E13), most cells of the
pancreatic anlage were Pref-1 positive, becoming predominantly
restricted to the insulin-producing cells during development. In
conclusion, these findings suggest that Pref-1 is involved in both
differentiation and growth of ß-cells. |
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Materials and Methods |
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RNA isolation
After isolation or culture, islets were washed in Hanks’
Balanced Salt Solution and immediately dissolved in RNAzol (Biotecx
Laboratories, Austin, TX). Embryonal and neonatal pancreas [embryonal
day 12 (E12) to postnatal day 4 (P4)] as well as adrenals,
pituitaries, and muscle from 3-month-old male Wistar rats were
dissected out, immediately washed in Hanks’ Balanced Salt Solution,
and homogenized in RNAzol. RNA was extracted and subsequently
precipitated by adding 0.2 M NaCl and 2 vol absolute
ethanol (-20 C). For mRNA isolation, the QuickPrep micro-mRNA
purification kit (Pharmacia LKB Biotechnology, Uppsala, Sweden) was
used. Briefly, islets were homogenized immediately in extraction
buffer, and mRNA was isolated with oligo(deoxythymidine)-cellulose and
precipitated as described above, with the addition of glycogen as
carrier.
Differential screening
Twenty thousand clones of a size-fractionated cDNA library
ligated into pCDM8 in MC1061/p3, made from neonatal Wistar rat islets
stimulated for 24 h with 1 µg/ml hGH, were plated and
subsequently transferred to nylon membranes. Filters were prehybridized
in 6 x SSC (0.90 M NaCl and 0.090 M
sodium citrate), 1% SDS, 10 x Denhardt’s reagent (10
mM EDTA, 100 µg/ml polyadenylase, 100 µg/ml yeast RNA,
and 100 µg/ml herring sperm DNA) at 68 C for 2–5 h (20).
Hybridization was performed under the same conditions but without
Denhardt’s reagent and with the addition of control islet cDNA (>700
bp) radioactively labeled using the Rapid Multiprime DNA Labeling Kit
(Amersham, Aylesbury, UK). Positive clones were identified after
exposure overnight to a PhosphorImager screen and using the
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). After removing
labeled DNA by boiling the membranes in NaOH, a second hybridization
was performed using a labeled probe made from hGH-stimulated islet cDNA
(>700 bp). Bacterial clones hybridizing with higher intensity to the
cDNA from hGH-treated islets than to cDNA from control islets were
amplified in culture, the cDNA insert was isolated and ligated into the
pGEM vector for sequencing using the AutoRead Sequencing kit
(Pharmacia) and the automated laser fluorescent DNA sequencer
(Pharmacia). Clones were subsequently analyzed by an identity search in
the program FASTA in GenBank.
Cloning of rat Pref-1
To obtain a full-length cDNA corresponding to the 600-bp
fragment identified from the first screening, primers spanning the
5'-end of Pref-1 were made for PCR using the mouse sequence as a
template (21). An 800-bp fragment of the 5'-end of rat Pref-1 was
amplified and cloned into the pCR vector using thymidine adenine
cloning (Invitrogen, Leek, The Netherlands). The full-length cDNA was
then obtained by PCR using the two plasmids containing the 3'-end and
the 5'-end, primers spanning the reading frame sequence, and subsequent
thymidine adenine cloning.
Northern blot analysis
Total RNA was incubated at 65 C for 15 min in 50% formamide and
35% formaldehyde in 1 x
3-(N-morpholino)propanesulfanic acid (MOPS). RNA was
size-fractionated in a 1% agarose gel containing 2.2 M
formaldehyde in 1 x MOPS buffer (20). Subsequently, the RNA was
transferred overnight to either nitrocellulose or nylon membranes in
20 x SSC. Prehybridization was performed for 3–6 h at 68 C in
100 µg/ml herring sperm DNA, 0.1% SDS, 5 x SSC, 5 x
Denhardt’s reagent, and 20 mM
Na3PO4 (pH 6.8). Filters were hybridized
overnight at 68 C in the same solution with the exception of 1 x
Denhardt’s reagent and addition of the extracellular part of Pref-1
(1–686 bp) radioactively labeled (
106 cpm/ml) using
random primer DNA labeling (Amersham). The filters were washed, exposed
to a PhosphorImager screen overnight and analyzed using the ImageQuant
program and the PhosphorImager. Filters were rehybridized to a rat
cyclophilin probe (a 477-bp cDNA AluI fragment) used as an
internal standard. Results were expressed as the percent Pref-1 mRNA of
cyclophilin mRNA.
Ribonuclease (RNase) protection assay (RPA)
As probes for RPA, a SalI fragment of the
intracellular part of Pref-1 (730–1446 bp) and a NcoI
fragment of cyclophilin cDNA (250 bp) were used. Labeling was performed
using 10 mM dithiothreitol, 1 U RNasin RNase inhibitor, 500
mM nucleotides (ATP, GTP, and CTP), 6.25 µM
cold UTP, 1 U SP6 polymerase, and 50 µCi [32P]UTP. RNA
was extracted with phenol-chloroform-isoamylalcohol (25:24:1,
vol/vol/vol) and precipitated. The specific activity of the probe was
determined by trichloroacetic acid precipitation. Total RNA (1–10
µg) was hybridized overnight with the labeled probes (SA, 100,000
cpm/sample) at 45 C as described in the RPA kit II (Ambion, Austin,
TX). Samples were digested with RNase and precipitated. After
size-fractionation on a 6% polyacrylamide gel, the protected bands
were analyzed as described above using the PhosphorImager. Pref-1 mRNA
was quantitated relative to the cyclophilin mRNA.
Quantitative RT-PCR
cDNA was synthesized from 1 µg total RNA from adult adrenals,
pituitaries, muscle, and islets from neonatal, adult, and pregnant
Wistar rats using a random primer. Primers were made to detect the
5'-end of Pref-1 (279–554 bp), and PCR was performed using 1% of the
cDNA and 10 pmol/primer in 1.5 mM MgCl2, 125
nM deoxy-NTPs, [33P]CTP, and 1 x PCR
buffer. After the addition of 1 U Taq polymerase, the PCR
reaction was run for 27 cycles (30 min at 94 C, 60 min at 55 C, and 90
min at 72 C). The amplified products were separated on a 6% denaturing
polyacrylamide gel and subsequently analyzed as described above using
the PhosphorImager. As internal controls, primers amplifying
glucose-6-phosphate dehydrogenase (G6PDH) and/or cyclophilin were used.
For quantitation, both amplicons were in the exponential amplification
phase.
Antibody production
The intracellular (1102–1425 bp) and extracellular (211–299
bp) regions of Pref-1 were subcloned into the pGEX-3x vector containing
the glutathione S-transferase (GST) gene. The fusion
proteins were synthesized and purified according to the GST Gene Fusion
System (Pharmacia Biotech). New Zealand White rabbits were primed with
1 mg protein in TBS (50 mM Tris, pH 7.4, and 150
mM NaCl) and 50% complete Freund’s adjuvant using
multiple intradermal injections. Every second week booster injections
containing 150 µg protein in TBS and 50% incomplete Freund’s
adjuvant were performed by a single injection in the neck. The
antiserum was screened using immunohistochemistry.
Immunocytochemistry
Fetal Wistar rat pancreas from E13–E20 (n = 20) and
neonatal rat pancreas from P1 and P6 (n = 4) were immersion-fixed
in 4% (wt/vol) paraformaldehyde in 0.1 M sodium phosphate
buffer (pH 7.4) overnight at 4 C. Adult Wistar rats (n = 5) were
asphyxiated with carbon dioxide and perfused via the heart with 5 ml
saline, followed by 50 ml 4% (wt/vol) paraformaldehyde (pH 7.4). The
pancreas were then immersion-fixed in the same fixative overnight at 4
C. All specimens were routinely embedded in paraffin.
Antigen retrieval of deparaffinized and hydrated 3- to 5-µm sections was performed in a Polar Patent (PP-780) precision pulsed laboratory microwave oven (Ax-Lab, Copenhagen, Denmark) at maximal effect (780 watts) in 0.1 M citrate buffer (pH 6.0) three times for 5 min each time, followed by 20 min at room temperature. The sections were then treated with 0.1 M periodic acid for 5 min to inhibit endogenous peroxidase activity. Quenching of free aldehyde groups were performed with 0.01% (wt/vol) sodium borotetrahydride for 2 min, followed by preblocking with 10% normal serum from the species producing the second antibody. The sections were reacted with polyclonal Pref-1 antisera (dilution, 1:300) overnight at 4 C. The site of antigen-antibody reaction was visualized with a biotinylated goat antirabbit (Ig) antibody (Dakopatts, Dako, Glostrup, Denmark) and peroxidase-conjugated streptavidin (Dakopatts) and developed in diaminobenzidine-H2O2 medium. Nuclei were counterstained with hematoxylin. Controls consisted of conventional staining controls (22) as well as preabsorptions of the polyclonal Pref-1 antiserum with the GST-fusion proteins against the intracellular or extracellular part of the Pref-1 protein. For triple immunofluorescence, the sections were first reacted with a guinea pig insulin antiserum (Dakopatts), followed by a species-specific fluorescein isothiocyanate (FITC)-labeled goat antiguinea pig (Ig) antibody (Jackson ImmunoResearch Laboratory, West Grove, PA). Subsequently, sections were microwaved as described above and incubated with a mixture of rabbit anti-Pref-1 antiserum and a mouse monoclonal antiglucagon antibody (Novoclone, Novo-Nordisk, Bagsvard, Denmark), followed by species-specific Texas Red-labeled donkey antirabbit Ig (Jackson ImmunoResearch Laboratory), biotinylated goat antimouse Ig (Dakopatts) serum, and 7-amino-4-methylcoumarin-3-acetic acid-N'-succinimide ester (AMCA)-conjugated streptavidin (Vector Laboratories, Burlingame, CA). Immunofluorescence specimens were examined in a Leica DMRB epiillumination microscope (Leica A/S, Herlev, Denmark), using AMCA-, FITC-, and Texas Red-selective filter blocks.
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Results |
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-subunit of the stimulatory G protein
(Gs), and Pref-1. A fragment of 600 bp of the
3'-end from Pref-1 was identified from the first screening. As no
full-length clones were observed among the 8 clones found by
rescreening 40,000 colonies of the library using the 600 bp as a probe,
primers spanning 800 bp of the 5'-end of Pref-1 were made for PCR using
the mouse sequence as a template (21). The full-length clone was
subsequently obtained using PCR with the 2 vectors containing the
cloned 600 bp and the PCR-amplified 800 bp as templates. Sequencing of
the full-length clone gave a 1600-bp long cDNA (Fig. 1
). The identity search in GenBank
revealed 96% identity to the mouse Pref-1 (21) and Dlk (23) and 86%
identity to the human sequences, Dlk and pG2 (24) (Fig. 1). An analysis
of protein sequences revealed another protein with close similarity to
the extracellular part of Pref-1, namely fetal antigen-1 (FA-1) (25, 26) (Fig. 1). The protein sequence of pG2 is not shown in the figure
because a possible sequencing error resulted in an altered reading
frame (27).
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). In addition, two less abundant mRNAs
of approximately 4 and 7 kb were detected in the adrenals, whereas the
islets expressed somewhat shorter forms of 3.7 and 6.2 kb (data not
shown). Islets from adult female and male rats had undetectable levels
of Pref-1 mRNA using up to 10 µg total RNA in RPA. However, by RT-PCR
using primers amplifying part of the 5'-end, we were able to detect
this mRNA at very low levels in adult islets (0.9 ± 0.1%
Pref-1/cyclophilin) compared with neonatal islets (68 ± 8%;
n = 4; data not shown). Islets from pregnant rats revealed
increased levels (7–100 fold Pref-1/G6PDH) compared with islets from
age-matched nonpregnant female rats (49 ± 16%; n = 6; Figs. 3 and 4). A
peak in Pref-1 mRNA levels was observed on gestational day 14 as well
as around term (Fig. 4; n = 3). For comparison, high levels of
Pref-1 mRNA were found in adrenals (328 ± 63% Pref-1/G6PDH) and
pituitaries (869 ± 156%) from adult rat (Fig. 3; n = 6).
Pref-1 mRNA was present in the pancreas throughout development from E12
to P4, with a 2-fold increase on E17 and a 5-fold increase at birth
compared with the level on E12, followed by a decrease around P4 (Fig. 5; n = 3; fetuses from three
pregnant rats), as determined by RPA. Examination of isolated islets
from E19 to P10, showed high levels of Pref-1 mRNA expression until day
P2, followed by a decrease (Fig. 6;
triplicates from fetuses of one pregnant rat).
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; n = 4). By RPA,
islets cultured for 24 h with hGH showed a 2-fold stimulation,
which increased to 5-fold after 48-h culture with hGH compared with
control islets (Fig. 7). The GH
concentration dependency of Pref-1 mRNA from neonatal islets cultured
for 3 days (0.1–2.0 µg/ml) was determined using RPA and produced a
bell-shaped curve, with a maximum at 0.5–1 µg/ml (Fig. 8). To test the hormonal specificity,
neonatal rat islets were cultured for 3 days with either hGH, bGH, or
oPRL, all of which were found to stimulate the levels of Pref-1 mRNA 3-
to 4-fold compared with levels in untreated control islets (Fig. 9; n = 3).
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, A and C). In contrast, islet-like
clusters of cells showed weak or no staining (Fig. 10A). With ensuing
development (E17–E19), Pref-1 immunoreactivity gradually disappeared
from the nonislet cell compartment of the pancreas. An increased
staining of the islets of Langerhans as well as of small islet-like
clusters was noted from E19 onward (Fig. 10F). In neonatal and adult
rats, Pref-1 immunoreactivity was virtually restricted to the islets of
Langerhans, small islet-like clusters, and solitary cells interspersed
in the exocrine compartment of the pancreas. The staining of the
endocrine cells was predominantly cytoplasmic and granular. In the
adult pancreas, however, the islet cells staining for Pref-1 appeared
very weak (data not shown). Using the antibody raised against the
extracellular domain of Pref-1, the same pattern was seen throughout
development of the pancreas (Fig. 10D). All controls appeared negative
(Fig. 10, B, E, and G). Triple immunofluorescence of E19 and neonatal
(P1) pancreas showed Pref-1 to be present in insulin-containing cells
(Fig. 11, A–D). However, not all
insulin-containing cells were immunopositive for Pref-1, and a few
Pref-1-immunopositive cells were only weakly positive for insulin. On
E19, scattered cells costoring insulin and glucagon were regularly
detected; however, these cells were rarely immunopositive for Pref-1.
The vast majority of glucagon-positive (but insulin-negative) cells
were Pref-1 negative, although a few (<1%) Pref-1-positive glucagon
cells were detected. All controls appeared negative.
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Discussion |
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Mouse Pref-1 has previously been shown to be a 7.3-kb long gene consisting of five exons and four introns (28). The protein has been described as a transmembrane protein of 383 amino acids, with a signal sequence and six epidermal growth factor (EGF)-like domains extracellularly (21). Pref-1 mRNA has previously been detected in mouse embryos from E8.5–E18.5. It was abundant in a 13-day-old embryo, where it was mainly present in pituitary, liver, lung, the mesenchymal part of the vertebrae, and the tongue (21). This is in contrast to adult tissues, where Pref-1, Dlk, and pG2 mRNA have been shown to be present exclusively in adrenals, placenta, and a number of neuroendocrine tumors (21, 23, 24). Pref-1 has been found to be down-regulated during adipocyte differentiation of the preadipocyte-like cell line 3T3-L1 and to prevent differentiation when expressed constitutively in these cells (16). In human adrenals, Pref-1 mRNA has been shown to be a midgestational marker of the chromaffin cell lineage and was found to increase 4 weeks after tyrosine hydroxylase; it is the first identified marker of chromaffin development (23). Amino acid sequence alignment revealed high similarity of Pref-1 to human FA-1, a protein with 225–262 amino acids that is found in human amniotic fluid during the second trimester of pregnancy (25). This protein, which corresponds to the extracellular part of Pref-1, has been shown to be present in fetal liver (29), adrenals, and pancreas as well as in adult adrenals, placenta, and islets of Langerhans, where it is colocalized with insulin in some of the granules in the ß-cells (26, 30, 31). A high concentration of FA-1 (20 µg/ml) has been found in fetal serum, whereas the concentration in normal serum was around 20 ng/ml (26). In the circulation of pregnant mice, high concentrations were also found, followed by a dramatic fall after delivery, suggesting that fetal and placental tissues are the major sources of FA-1 (32).
We found that the size of the rat Pref-1 transcript from islets of Langerhans was 1.6 kb, as previously found in mouse adrenals and preadipocytes (16, 17). However, two additional, less abundant mRNAs, approximately 4 and 7 kb in adrenals and 3.7 and 6.2 kb in islets, were identified, indicating that Pref-1 mRNA may exist as splice variants and also that the protein may be differentially expressed in a tissue-specific manner. The amount of Pref-1 mRNA in adult islets was very low and only detectable by RT-PCR. Interestingly, in islets from rats on day 12 of pregnancy to 4 days after term Pref-1 mRNA was shown to be up-regulated compared with that in islets from age-matched female rats. This corresponds well to the ß-cell hyperplasia seen during pregnancy (33). The increase in Pref-1 mRNA expression on day 14 of pregnancy actually coincides with the time that the replication rate of the ß-cells from pregnant rats has been found to be at the highest level (34). At this time point also the major lactogenic hormone during pregnancy, placental lactogen, is as high as at term, which might influence the second peak of Pref-1 mRNA. The replication rate in the pregnant rat returns to normal on day 20, probably due to counterregulatory mechanisms, for instance the high levels of steroids as suggested by Parsons et al. (34).
During the embryonic development of the rat pancreas an increase in Pref-1 mRNA levels was detected from E13–E17. Furthermore, a marked increase was observed just before birth, followed by a drastic decline a few days after birth, to similar levels found in adult islets. When comparing data from total pancreas and isolated fetal and neonatal islets, an apparent low amount of Pref-1 mRNA was found in islets. If Pref-1 acts as an adhesion molecule, it could be due to the collagenase isolation method used for the islets. This might result in a subpopulation of islets containing the least Pref-1.
A dramatic change in the Pref-1 expression pattern occurs during development, from being present in most of the nonislet cells until day E19, to a situation where Pref-1 immunoreactants become virtually restricted to the insulin-containing cells. A similar change in expression pattern for FA-1 has been observed in human fetal pancreas (30, 35), where FA-1 was shown to exclusively colocalize with the insulin-producing endocrine cells throughout development, whereas it was absent from glucagon-expressing cells in both the developing pancreas and in neoplasms (35). This pattern was not observed in the rat pancreas, as we occasionally detected endocrine cells coexpressing insulin and glucagon to be weakly Pref-1 immunoreactive. Additionally, a minority (<1%) of Pref-1-positive cells were immunoreactive for glucagon, although the vast majority of Pref-1-positive cells coexpressed insulin. It is striking that the homeo-box containing transcription factor PDX-1 (IDX-1, Ipf-1, and Stf-1) also shows a similar expression pattern, i.e. present in all of the epithelial cells in the early pancreas, whereas the later is virtually restricted to the ß-cells (36).
The role of Pref-1/FA-1 in ß-cell development and function is still
obscure. As the EGF repeats of Pref-1 have high identity to the EGF
repeats of the Drosophila protein 
(17) (indicating no
binding to the EGF receptor), it has been proposed that Pref-1, in
analogy with , participates in cell-cell interaction by binding to a
notch-like receptor protein with signaling properties maintaining the
early embryonic pancreas in a proliferative, but less differentiated,
state (37). It may be speculated that the expression declines in the
majority of the pancreatic parenchymal cells and becomes virtually
restricted to the ß-cells, thereby allowing the differentiation of
the exocrine cells to proceed while the proliferation of ß-cells,
which is at its maximum around birth (38), is maintained by the
presence of Pref-1, as proliferation requires that the ß-cells be in
an immature state. It may be of significance that the down-regulation
of Pref-1 in the ß-cells shortly after birth coincides with the gain
in glucose sensitivity of the insulin secretory apparatus (39). The
increased level of Pref-1 mRNA in islets during pregnancy is somewhat
more puzzling, but might suggest that during the increased growth and
differentiation of the ß-cells, a transitional state of more immature
ß-cells is present. Interestingly, islet cells in monolayer culture
with hGH respond poorly to an acute glucose challenge, thus resembling
the fetal ß-cells (Nielsen, J. H., unpublished observation).
Also, after the first 1–2 weeks following birth, the rate of ß-cell
proliferation is markedly reduced (40), as is the expression of
insulin-like growth factor II (41) and TRH (42), suggesting a
transition from an immature proliferative state to a mature, terminally
differentiated ß-cell. This transition may be associated with a
concomitant loss of immature cells by apoptosis, as recently
demonstrated in islets of 2-week-old rats (40).
The up-regulation of Pref-1 expression in islets by GH and PRL is in
accordance with its increased expression in islets during pregnancy,
supporting a permissive role of Pref-1 in cell replication. The finding
that both bGH and oPRL induced Pref-1 mRNA expression indicates that
Pref-1 is regulated by both GH and PRL receptors activated by hGH. The
maximal stimulating concentration of hGH was about 0.5–1 µg/ml, and
the bell-shaped dose-response curve is in accordance with other effects
of GH, reflecting activation of the GH receptor by dimerization (43).
It was recently shown that GH stimulates insulin gene transcription by
activation of STAT5 (signal transducer and activator of transcription)
binding to an -activated sequence-like element in the 5'-flanking
region (44). Whether the stimulation of Pref-1 expression is a result
of direct transcriptional activation via the JAK/STAT pathway remains
to be determined. In conclusion, these results show that Pref-1
expression is high in islets from newborn and pregnant rats as well as
in the early embryonic pancreas in vivo. Furthermore, we
have demonstrated that Pref-1 mRNA expression is up-regulated by GH and
PRL in rat pancreatic islets in vitro, suggesting that
Pref-1 plays a role in the differentiation and proliferation of the
pancreatic ß-cell.
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Acknowledgments |
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Footnotes |
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Received January 31, 1997.
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Y. S. Moon, C. M. Smas, K. Lee, J. A. Villena, K.-H. Kim, E. J. Yun, and H. S. Sul Mice Lacking Paternally Expressed Pref-1/Dlk1 Display Growth Retardation and Accelerated Adiposity Mol. Cell. Biol., August 1, 2002; 22(15): 5585 - 5592. [Abstract] [Full Text] [PDF]
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R. Graichen, D. Liu, Y. Sun, K.-O. Lee, and P. E. Lobie Autocrine Human Growth Hormone Inhibits Placental Transforming Growth Factor-beta Gene Transcription to Prevent Apoptosis and Allow Cell Cycle Progression of Human Mammary Carcinoma Cells J. Biol. Chem., July 12, 2002; 277(29): 26662 - 26672. [Abstract] [Full Text] [PDF]
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M. Andersen, C. H. Jensen, R. K. Stoving, J. B. Larsen, H. D. Schroder, B. Teisner, and C. Hagen Fetal Antigen 1 in Healthy Adults and Patients with Pituitary Disease: Relation to Physiological, Pathological, and Pharmacological GH Levels J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5465 - 5470. [Abstract] [Full Text] [PDF]
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 D. Le Roith, C. Bondy, S. Yakar, J.-L. Liu, and A. Butler The Somatomedin Hypothesis: 2001 Endocr. Rev., February 1, 2001; 22(1): 53 - 74. [Abstract] [Full Text]
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 A. A. Wylie, S. K. Murphy, T. C. Orton, and R. L. Jirtle Novel Imprinted DLK1/GTL2 Domain on Human Chromosome 14 Contains Motifs that Mimic Those Implicated in IGF2/H19 Regulation Genome Res., November 1, 2000; 10(11): 1711 - 1718. [Abstract] [Full Text]
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