| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Instituto de Investigaciones Biomédicas "Alberto Sols", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid (A.L., P.S.), 28029 Madrid; and Servicio de Cirugía Pediátrica, Hospital Infantil La Paz, (A.L., J.A.T., H.M.X., J.A.D-P), 28046 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Pilar Santisteban, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain. E-mail: psantisteban{at}iib uam.es.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and HNF-3ß
(hepatocyte nuclear factors 3 and ß), the homeodomain proteins
Hoxa5 and TTF-1 (thyroid transcription factor 1), and members of the
nuclear receptor superfamily (including receptors for glucocorticoids,
thyroid hormones, and retinoids) are involved. TTF-1 is a
homeodomain-containing transcription factor (2), expressed in two of
the many structures derived from the foregut endoderm, the thyroid, and
the lung (3). TTF-1 messenger RNA (mRNA) is detectable within the
ventral migrating edge of the lung bud on embryonic day (E) 9.5 in the
rat (3). In E11.5, a strong signal can be detected in both branches of
the primitive bronchi, and from E13.5–E15.5, TTF-1 mRNA is expressed
in the bronchial epithelium. In late gestation stages (E17.5), TTF-1 is
present in epithelial cells of the bronchioli. TTF-1 is involved in the
transcription of pulmonary surfactant proteins (SP-A, SP-B, and SP-C)
and clara cell secretory protein (4, 5, 6, 7), as well as in
thyroid-specific genes such as thyroglobulin, thyroid peroxidase, TSH
receptor, and Na+/I-
symporter (8, 9, 10, 11, 12, 13, 14). Surfactant protein B (SP-B) is a hydrophobic protein
that enhances the spreading and stability of surfactant phospholipids
in the alveolus. Ablation of the SP-B gene in transgenic mice
demonstrates the critical role of SP-B in the initiation of lung
inflation at birth. Homozygous SP-B (-/-) mice died of respiratory
failure immediately after birth (15), and their lungs, normally
developed, remained atelectatic. An essential role for TTF-1 in
lung and thyroid morphogenesis has been established, because homozygous
TTF-1 null mutant mice show dilated sac-like structures in the pleural
cavity rather than normal lungs (16). These structures had a
rudimentary bronchial tree (but no bronchioli, alveoli, or pulmonary
parenchyma) and an abnormal epithelium. This mutant died of respiratory
distress immediately after birth and lacked the thyroid; the
hypothalamus was severely affected (16) and have defects in
tracheoesophageal morphogenesis (17). Other transcription factors are
required for normal lung morphogenesis, such as HNF-3 and HNF-3ß,
both of which are expressed in endoderm derivatives during
embryogenesis and lung development (18, 19) and are involved in
regulation of TTF-1 gene expression (20). The role during
development of the glucocorticoid receptor (GR) was investigated by
generation of GR-deficient mice (21). GR null mice show respiratory
distress at birth and die within a few hours. The lungs are severely
atelectatic, and development is impaired from day 15.5 of gestation.
These observations suggest: 1) a possible role for these transcription
factors in the pathogenesis of pulmonary hypoplasia; and 2) prompt
research into how their expression is altered in this disorder during
embryogenesis. Newborns with congenital diaphragmatic hernia, a malformation that occurs in up to 1:2200 births, still have a poor prognosis, in spite of recent therapeutic progress. This high mortality rate is attributable mainly to neonatal respiratory failure caused by the associated severe pulmonary hypoplasia, with physiological and biochemical immaturity. Aiming at better understanding the pathophysiology of pulmonary hypoplasia, a reproducible animal model was developed in neonatal rats by feeding nitrofen (NF) (2,4-dichlorophenyl-p-nitrophenyl ether) to timed-mated pregnant rats in an appropriate embryological window (22). Lung hypoplasia (23) and immaturity (24), observed in rat fetuses born to exposed females, are very similar to those found in the human disorders. In this rat model, the most severe pulmonary defects were observed only after exposure to NF between E9–E11, and not before this stage (22). Nitrofen is detected in the embryonic compartment, 3 h after administration, with a peak, 72 h later, that corresponds to E12–E14 (25). Nitrofen is thus present in the embryo at the time at which TTF-1 is expressed in lung. These observations suggest a possible alteration of TTF-1 in lung hypoplasia. Using the above rat model, we determined TTF-1 expression as a marker of adequate lung morphogenesis, and SP-B expression as an indirect marker of lung maturity. We report here that expression of TTF-1 is severely decreased in lung hypoplasia.
Glucocorticoids are used clinically to induce lung maturation and surfactant production in human fetuses expected to be delivered prematurely. This antenatal hormonal therapy is known to accelerate pulmonary development in premature neonates and has decreased the incidence of respiratory distress syndrome and pulmonary complications of preterm birth (26, 27), but the mechanism remains unclear. The addition of TRH therapy is no more beneficial than glucocorticoids alone (28). We investigated the effect of this antenatal hormonal therapy on TTF-1 expression. Our results show that glucocorticoid therapy to animals with lung hypoplasia restores the expression of this transcription factor, supporting routine in utero glucocorticoid treatment for patients expected to suffer lung hypoplasia.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Experimental design
A scheme representing the animal treatment appears in Fig. 1
and was carried out as follows. After
light ether anesthesia, time-mated pregnant rats received
intragastrically, on day 9.5 of gestation, either 100 mg NF (NF group)
(Wako Chemicals, Neuss, Germany) diluted in pure olive oil (1)
or the same volume of vehicle (control group). Dams of both groups also
received different hormonal therapies to accelerate pulmonary
development. The dexamethasone (Dex) and NF + Dex groups received 0.4
mg/kg Dex ip on days 19 and 20. The TRH and NF + TRH groups were
implanted ip on day 18 with an osmotic minipump (Alzet, model 2ML2;
Alza Corp., Palo Alto, CA) containing 100 µg/kg TRH,
after which 25 µg/kg TRH were ip injected. Other groups of animals
were treated with combined hormonal treatment (Dex + TRH and NF + Dex +
TRH groups), receiving Dex and TRH as described for Dex and TRH groups.
To reproduce all manipulations, animals not treated with TRH had an
Alzet minipump containing saline buffer, and animals not treated with
Dex received ip saline buffer. TRH and Dex were purchased from
Sigma (St. Louis, MO). The fetuses were recovered on day
21 by cesarean section, examined, weighed, and dissected under a
binocular operating microscope for diaphragm inspection and lung
recovery. Rat fetuses were weighed and their lungs immediately frozen
for subsequent RNA and nuclear protein extraction. Fetal lung weight/BW
ratio (mg/g) was significantly smaller (P < 0.05) in
NF-treated rats (16.68 ± 1.02), compared with control groups
(31.33 ± 1.15), indicating that NF clearly produces lung
hypoplasia, thus validating our model of study. Dex reverts the effect
of NF on lung hypoplasia (24.05 ± 1.95), making its effect
statistically significant (P < 0.05).
|
RNA isolation and Northern blot analysis.
Fetal lungs from different groups were pooled separately, and
total RNA was isolated with guanidine isothiocyanate and cesium
chloride centrifugation (32). Total RNA was electrophoresed on a
formaldehyde-1% agarose gel and transferred to nytran membrane
(Schleicher & Schuell, Inc., Keene, NH). Hybridization was
carried out using a 0.6-kb EcoRI fragment of rat TTF-1 (2)
and a 0.5-kb PstI fragment of rat SP-B (33). All probes were
isolated and labeled with [-32P]
deoxycytidine triphosphate (ICN Biochemicals,
Irvine, CA.) by random oligopriming to a specific activity of 1 x
109 cpm/DNA. Northern blot images were analyzed
quantitatively using a scanning densitometer and image software and
normalized with the methylene blue-stained 18S ribosomal RNA.
Transfection assays
Ten micrograms of a luciferase-linked chimeric construct,
spanning 2.8 kb of the human TTF-1 5'-flanking region subcloned in the
reporter vector pSV0AL-A
5' (34), were transiently transfected into
H-441 cells by the calcium phosphate precipitation method (35). The
plasmid RSV-CAT (2 µg) (36) was also transfected and used to
normalize transfection efficiency. After transfection, cells maintained
under different conditions were harvested. Luciferase and CAT
activities were measured as described (35, 37). In the experiments
indicated, 2 µg of the TTF-1 expression vector (pCMV-THA) (38) or the
corresponding empty vector (pRC-CMV) were cotransfected.
Electrophoretic mobility shift assays
Nuclear extracts from lungs were prepared, following the method
of Gorski et al. (39). Protein concentration was determined
with the Bio-Rad Laboratories, Inc. kit (Bio-Rad Laboratories, Inc., Richmond, CA) and BSA as standard. Gel shift
assays were performed with the TTF-1-binding site of human SP-B
promoter as probe (4). The oligonucleotide SPB-f1, derived from
positions -113 to -90 of human SP-B promoter, was labeled with
T4 polynucleotide kinase and
[
-32P] ATP (ICN., Irvine, CA) and annealed
as described (40). For binding reactions, 10 µg of nuclear proteins
were preincubated in a binding reaction mixture containing 40
mM HEPES (pH 7.9), 200 mM
KCl, 0.5 mM dithiothreitol, 0.2
mM EDTA, 5% Ficoll, and 3 µg poly (dI-dC) for
15 min on ice. In competition experiments, the unlabeled
oligonucleotide was added in excess (100x). Labeled oligonucleotide
was added to the mixture and incubated for 30 min at room temperature.
For supershift assay, 1 µl anti-TTF-1 antibody (3) was added before
the addition of the probe and was incubated for 3 h. As a control
of supershift specificity, a preimmune serum was used and incubated in
the same conditions. The resulting DNA-protein complexes were separated
from free DNA on a 5% polyacrylamide gel (29:1,
acrylamide-bisacrylamide). Gels were resolved at 20 mA in a cold room
in 0.5 x TBE [1 x TBE is 90 mM Tris,
90 mM boric acid, and 1 mM
EDTA (pH 8)] before being vacuum dried and exposed to x-ray film at
-70 C.
Immunoblotting analysis
Nuclear protein extracts (40 µg) were separated in SDS-PAGE.
Proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Inc.), in a buffer containing 25 mM Tris,
200 mM glycine, and 20% methanol. After blocking the
membranes with 10% low-fat dried milk in Tris-buffered saline
containing 0.05% Tween-20, immunodetection was performed using the
antibody anti-TTF-1 (3). After probing with the antibodies, membranes
were incubated with a streptavidin-conjugated antirabbit-specific
secondary antibody. Immunoreactive bands were visualized using the
luminol reagent (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA). To assess specificity and equivalent loading of the samples,
the same blots were reprobed with a specific anticonstitutive
transcription factor anti-CTF/NF-1 antibody (sc-870) (Santa Cruz Biotechnology, Inc.).
Statistical analysis
Statistical significance between different treatments was
determined using Student’s t test. Differences are
considered significant at P < 0.01.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
The values obtained after quantitation were 75% lower than the
corresponding control (Fig. 2C, compare Control and NF groups). Based
on the extensively use of Dex as a treatment to induce lung maturation,
we next studied its effect in TTF-1 mRNA levels of control and
hypoplastic fetal lungs. Pregnant rats, untreated or treated with NF,
received Dex, as described in Materials and Methods. Total
RNA was extracted from fetal lungs, and the TTF-1 mRNA levels were
analyzed by Northern blot. Interestingly, Dex treatment markedly
increased (10-fold) TTF-1 mRNA levels when it was administered to
NF-treated rats (Fig. 2, A and C, compare NF with NF + Dex groups), but
it had no significant effect on control rats (Fig. 2, A and C, compare
Control with Dex groups). The observation that Dex treatment of
hypoplastic lungs not only rescues the TTF-1 mRNA levels but also
increases these levels over control values (3-fold) (Fig. 2, A and C,
compare Control with NF + Dex groups) is very important. It has been
suggested that combined antenatal glucocorticoid and TRH treatment
result in synergistic improvement of lung function of premature
neonates (41). We studied the role of this combined treatment in lung
hypoplasia. TRH was administered alone or with Dex to pregnant mothers,
untreated or previously treated with NF, as described in
Materials and Methods. The results obtained in Northern blot
analysis show that TRH alone increases TTF-1 mRNA levels (5-fold) when
given to NF-treated rats (Fig. 2, A and C, compare NF and NF + TRH
groups), but it has no a significant effect when given to control rats
(Fig. 2, A and C, compare Control and TRH groups). It is important to
mention that the increase obtained with TRH was approximately half of
that obtained with Dex and that, when both hormones were coadministered
to NF rats, TRH decreased by 2-fold the marked effect obtained with Dex
on TTF-1 mRNA levels (Fig. 2, A and C, compare NF + Dex with NF + Dex +
TRH groups). The combined Dex and TRH treatment did not induce a
significative effect on TTF-1 mRNA levels when administered to control
rats (Fig. 2, A and C, compare Control and Dex + TRH groups).
|
, B and D). The hormone therapy has an action on the SP-B mRNA
levels similar to that obtained for TTF-1 transcript, although some
differences have been observed. Thus, Dex markedly increased SP-B mRNA
levels, both in NF-treated (18-fold) and control (7-fold) rats (Fig. 2, B and D). TRH, alone or combined with Dex, increased SP-B mRNA levels
more than TTF-1 mRNA levels, although to a lesser extent than the
glucocorticoid alone. The effect was observed in both NF-treated and
untreated (control) rats. It is important to comment on two
observations: 1) the maximum effect on SP-B mRNA levels was obtained in
hypoplastic lungs treated with Dex, with increases of 7- to 8-fold over
control values; and 2) the TRH effect counteracts the Dex effect when
administered to hypoplastic lungs. From these data, we conclude that
TTF-1 and SP-B are strongly decreased in lung hypoplasia. Furthermore,
SP-B is under hormonal regulation in both normal and hypoplastic lungs,
whereas TTF-1 is regulated only in hypoplastic lungs. These results
suggest that SP-B is regulated by TTF-1-dependent and -independent
mechanisms and that TTF-1-mediated regulation is an important mechanism
of control in lung hypoplasia.
Because Dex treatment induces the maximal effect on TTF-1 mRNA levels,
when administered to rats with hypoplastic lungs, we next determined
whether this effect is correlated with protein levels. We assayed TTF-1
protein, by immunoblotting analysis, in nuclear protein extracts (Fig. 3) and found that TTF-1 protein levels
are drastically reduced in hypoplastic lungs (NF), as compared with the
control group. Again, Dex has no effect when administered to control
animals (Dex), but it increased TTF-1 protein levels, over the control,
when administered to NF-treated rats (NF + Dex). The effect is
specific, as demonstrated by reprobing the same Western blots with the
constitutive CTF/NF-1 transcription factor, also expressed in lungs
(42). These results suggest that lung hypoplasia is caused by
down-regulation of the homeotic transcription factor TTF-1. The
possible mechanism of the therapeutic action of glucocorticoids in lung
hypoplasia, inducing lung maturation and surfactant production, may be
explained, at least in part, by an increase in TTF-1 gene
expression.
|
show a retarded
protein/DNA complex in the control groups (lane 2), which is
consistently reduced in NF-treated rats (lane 8). The complex is
specific, as demonstrated in competition experiments with a 100-fold
excess of a related (lane 3) or an unrelated (lane 4) oligonucleotide.
We also investigated the effect of hormonal therapy in the gel shift
assay. Dex, TRH, or both hormones together did not modify the intensity
of the DNA complex when nuclear extracts from control groups were
studied (Fig. 4, compare lane 2 with lanes 5–7). Interestingly, the
administration of Dex to NF-treated rats showed a marked increase in
the intensity of the complex (Fig. 4, compare lanes 8 and 9), whereas
TRH (lane 10) or TRH plus Dex (lane 11) increased it only slightly.
Supershift assays were performed to confirm TTF-1 implication in
complex decrease or increase (Fig. 5).
The complex formed with nuclear extract from control lungs (lane 2) was
recognized by a specific -TTF-1 antibody inducing a clear supershift
(lane 5). The protein/DNA (lane 6) and TTF-1 supershift (lane 8)
complexes decreased in NF-treated rats; and again, Dex restored both
complexes (lane 9 and 11) over control levels when administered to
hypoplastic lungs. The specificity of the complex was assayed with the
corresponding 100-fold excess of the related (lanes 3, 7, and 10) and
unrelated (lane 4) oligonucleotides. As a control of the supershift
assay, the labeled oligonucleotide was preincubated with the -TTF-1
antibody alone, but this failed to produce the supershifted band (lane
12). Furthermore, the incubation with a preimmune serum did not
induce a supershift in control nuclear extracts (lanes 14 and
15). From these results, we conclude that the variation observed on
TTF-1 mRNA levels in all the experimental approaches studied in this
work parallels the variation found in TTF-1 protein levels and in its
binding activity.
|
|
), was transiently transfected into
H441 cells. In all transfections, the reporter vector pSV0AL-A5 (34)
was transfected in parallel as control. After transfection, cells were
maintained in medium containing only 0.2% FBS, with or without 1.5
µM NF, for the following 48 h. The low serum was
used to detect the Dex effect more clearly; as in 10% serum, the
hormone effect is masked by the endogenous glucocorticoid content of
serum (45). The cells were then treated, or not, for another 24 h
with 100 nM Dex. Nitrofen decreased TTF-1 promoter activity
approximately 3-fold, relative to control cells (Fig. 6B). Dex
treatment increased the promoter activity in NF-treated cells but not
in control cells.
|
), because of the
existence of multiple TTF-1-binding sites in this promoter (Fig. 6A)
(34). The down-regulation of TTF-1 elicited by NF, as well as the
reestablishment of activity induced by Dex, also occurred when the
exogenous transcription factor was cotransfected (Fig. 6C). These data
suggest that NF may act through a mechanism that impairs the
transcriptional activity of TTF-1 and that the glucocorticoids would
counteract this mechanism. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Using the above model, we demonstrate that the homeodomain transcription factor TTF-1 has an essential role in the pathogenesis of lung hypoplasia (16, 29). This role has been also demonstrated in mice lacking TTF-1 expression. Mutant Hoxa-5 mice (46) also have defects in lung ontogeny caused by reduction of TTF-1 expression, inducing a significant decrease in SP proteins that cause respiratory dysfunction similar to the respiratory distress syndrome described in premature human infants. Our results show that hypoplastic lungs have severely decreased TTF-1, mRNA, and protein levels and a consequential reduction in TTF-1-binding activity to the proximal SP-B promoter. Moreover, the upstream DNA of the rat and human TTF-1 gene possesses many putative TTF-1-binding sites (34, 47), suggesting that TTF-1 gene may be autoregulated (34). In consequence, a reduction in TTF-1 binding to its own promoter should have an effect at the transcriptional level. We have shown that NF, the herbicide that induces lung hypoplasia in rats, down-regulates the 2.8-kb fragment of the TTF-1 promoter activity, which includes the putative TTF-1-binding sites. In consequence, less transcription factor is synthesized. These results support the possible autoregulation of TTF-1, confirming that TTF-1 is a transcription factor essential in lung formation, and that its low expression is essential in the pathogenesis of lung hypoplasia.
Prenatal glucocorticoid therapy accelerates pulmonary development in premature infants (26, 27), and the addition of prenatal TRH therapy is no more beneficial than the use of glucocorticoids alone (28). Our results show that glucocorticoid treatment induces expression of TTF-1 mRNA and TTF-1 protein and, in consequence, induces an increase in TTF-1-binding activity to the SP-B proximal promoter, only in hypoplastic lungs. This increase is reproduced in TTF-1 promoter activity, although only in NF-treated cells. Glucocorticoids may thus restore TTF-1 and SP-B levels by increasing the TTF-1 autoregulatory mechanism. As a consequence, there is an increase in the TTF-1 protein level, which may then bind at a high ratio to its target promoter genes. We also have shown that treatment with TRH alone, or combined with Dex, had minimal consequences, as compared with the Dex effect in hypoplastic fetal lungs. These results are in agreement with a previous work that has shown that prenatal corticoid therapy improves pulmonary morphology in NF-treated rats, whereas TRH had minimal beneficial effects (48). Results also are in accordance with data on prevention of lung disease in human preterm infants (26).
Because NF induces a delay in lung development, the morphology of lungs from NF-treated fetuses is different from that of control, having proportionately more mesenchimal than epithelial cells (49). We believe that the effect of NF in fetal lung takes place mainly in epithelial cells affecting directly lung-specific genes expression. This idea is reinforced by the fact that the same results obtained in rats were found in epithelial culture of H441 cells. The intrinsic molecular mechanism of NF and glucocorticoids on TTF-1 gene expression is unknown. TTF-1 regulation has been extensively studied in FRTL-5 cells, showing down-regulation by TSH via cAMP (13, 50). To our knowledge, however, there is no evidence that NF increases cAMP in lung cells. Interestingly, clear down-regulation of TTF-1-binding activity is induced by an oxidative state (51), and reduction/oxidation (redox) reactions are intimately involved in the control of biological processes, including modulation of transcription factor function, e.g. AP-1 and NF-kB (52, 53). Antioxidant compounds are also described to reverse fetal rat lung hypoplasia (54), and Dex increases antioxidant enzymes (55). In addition, no functional glucocorticoid response elements have been reported in the promoter region of TTF-1. It thus seems that glucocorticoid response element-mediated gene induction is not the basis of this regulation. Based on these observations, we hypothesized that a possible mechanism for down-regulation of TTF-1 by NF and its restoration by glucocorticoids may be elicited by redox mechanisms. This idea is supported by the fact that cellular glucocorticoid responsiveness is coordinately modulated by the redox state and thioredoxin levels (56). This plausible mechanism remains to be studied; however, our data, showing that activity of exogenous TTF-1 cotransfected to H441 cells is also regulated by both NF and glucocorticoids, reinforce this hypothesis.
In conclusion, the data reported in this study demonstrate that lung hypoplasia is induced, at least in part, by an alteration of one of the main transcription factors involved in lung organogenesis. This work also supports routine in utero glucocorticoid treatment for patients expected to have lung hypoplasia.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Recipient of a fellowship from the Spanish Ministerio de
Educación y Cultura.
Received December 28, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, ß and genes in determination of the
definitive endoderm, chordamesoderm and neuroectoderm. Development 119:567–578
B. Involvement of a cellular reducing
catalyst thioredoxin. J Biol Chem 268:11380–11388This article has been cited by other articles:
 |
|
 |
|
  O. Boucherat, A. Benachi, A.-M. Barlier-Mur, M.-L. Franco-Montoya, J. Martinovic, B. Thebaud, B. Chailley-Heu, and J. R. Bourbon Decreased Lung Fibroblast Growth Factor 18 and Elastin in Human Congenital Diaphragmatic Hernia and Animal Models Am. J. Respir. Crit. Care Med., May 15, 2007; 175(10): 1066 - 1077. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Kolla, L. W. Gonzales, J. Gonzales, P. Wang, S. Angampalli, S. I. Feinstein, and P. L. Ballard Thyroid Transcription Factor in Differentiating Type II Cells: Regulation, Isoforms, and Target Genes Am. J. Respir. Cell Mol. Biol., February 1, 2007; 36(2): 213 - 225. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Das and V. Boggaram Proteasome dysfunction inhibits surfactant protein gene expression in lung epithelial cells: mechanism of inhibition of SP-B gene expression Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L74 - L84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. J. Pua, B. S. Stonestreet, A. Cullen, A. Shahsafaei, G. B. Sadowska, and M. E. Sunday Histochemical Analyses of Altered Fetal Lung Development Following Single vs Multiple Courses of Antenatal Steroids J. Histochem. Cytochem., December 1, 2005; 53(12): 1469 - 1479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Chapin, R. Ertsey, J. Yoshizawa, A. Hara, L. Sbragia, J. J. Greer, and J. A. Kitterman Congenital diaphragmatic hernia, tracheal occlusion, thyroid transcription factor-1, and fetal pulmonary epithelial maturation Am J Physiol Lung Cell Mol Physiol, July 1, 2005; 289(1): L44 - L52. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Oden and I. M. Cheifetz Neonatal Thyrotoxicosis and Persistent Pulmonary Hypertension Necessitating Extracorporeal Life Support Pediatrics, January 1, 2005; 115(1): e105 - e108. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. O. Nasonkin, R. D. Ward, L. T. Raetzman, A. F. Seasholtz, T. L. Saunders, P. J. Gillespie, and S. A. Camper Pituitary hypoplasia and respiratory distress syndrome in Prop1 knockout mice Hum. Mol. Genet., November 15, 2004; 13(22): 2727 - 2735. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
