1 Lung Development Research Program, Section of Pediatric Surgery, Department of Surgery, Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; and 2 Department of Surgery, Stanford University School of Medicine, Stanford, California 94305
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ABSTRACT |
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We have shown that dexamethasone (Dex) accelerates maturation and differentiation of cultured fetal murine lungs (Cilley RE, Zgleszewski SE, Krummel TM, and Chinoy MR. Surg Forum 47: 692-695, 1996). We now demonstrate that although Dex inhibits thinning of acinar walls and secondary septa formation, it does, however, promote lung growth. CD-1 murine fetal lungs were cultured for 7 days in the presence and absence of 10 nM Dex. Dex-modulated genes were investigated and identified by differential display of mRNAs performed with specific anchor primer H-T11G and 24 arbitrary primers. Thirty-five differentially expressed cDNAs were isolated, subcloned, sequenced, and identified through BLAST searches. One of these cDNAs, termed Dex2, with enhanced expression in Dex-treated lungs, had 100% similarity with ras-recision gene (rrg), also known as the lysyl oxidase (LOX) gene that encodes lysyl oxidase. LOX gene is very highly conserved, with significant sequence similarity among mouse, rat, and human. Two other cDNAs, termed Dex1 and Dex4, were also identified as rrg, with 92 and 97% sequence similarity with the existing data bank sequence of rrg. LOX enzyme is known to downregulate p21ras protein and play a central role in the maturation of collagen and elastin in the extracellular matrix as well as modulate the cytoskeletal elements. Thus LOX may be important in lung developmental processes involving epithelial-mesenchymal interactions.
lysyl oxidase; differential display of genes; epithelial-mesenchymal interactions; p21ras protein; lung development
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INTRODUCTION |
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THE REGULATION OF LUNG DEVELOPMENT is a complex process. A wide variety of genes and their products have been studied to increase understanding of their regulatory influences on maturation and differentiation of the developing lung. However, to date, the exact molecular mechanisms involved in the regulatory processes of lung development are not known.
Glucocorticoids have been shown to accelerate fetal lung type II cell maturation, which is mediated in part via fibroblasts. It is also known that dexamethasone (Dex), a synthetic steroid, promotes lung maturation both in vitro and in vivo. Similarly, it has been previously demonstrated that Dex enhances lung maturational processes in murine fetal whole lungs in culture (5, 8). Identifying and understanding the exact molecular pathways by which Dex carries out its effects may help in the design of new treatments for abnormally developing lungs, i.e., hypoplastic lungs. We propose that this lung organ culture model is useful for studying the isolated effects of Dex or other factors and thus helps identify the specific pathways involved in lung development.
In the present study, we employed the differential display technique to find differences in gene expression between untreated murine fetal lungs in culture (control) and those cultured in the presence of Dex. We isolated and identified the differentially expressed genes in these lungs by sequence similarity matches to existing genes in the GenBank database. Identifying the genes involved in lung developmental processes will open avenues for designing functional assays. We have standardized the technique of differential display of mRNAs in our laboratory, and, using this technique, our laboratory (4, 28) has previously demonstrated differential expression of genes between normal and nitrofen-induced hypoplastic murine fetal lungs.
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METHODS |
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Animals
Time-dated pregnant CD-1 mice were euthanized via halothane overdose on gestational day (GD) 14. Fetuses were harvested via laparotomy and placed in cold saline (on ice). Median sternotomy was performed under a dissecting stereomicroscope, and heart-lung units were excised with the trachea and larynx intact. The hearts were removed and the tracheae were transected. Isolated murine fetal lungs were cultured for 7 days.Culture Techniques
GD14 pseudoglandular lungs were placed individually onto 5-mm2 sterile membranes (0.45-µm pore size) that rested on sterile stainless steel wire mesh screens suspended in an organ culture dish filled with BGJb culture medium. Culture medium was supplemented with 100 U/ml of penicillin G, 0.1 mg/ml of streptomycin, 0.25 mg/ml of amphotericin B, and 1 mg/l of sodium ascorbate at pH 7.4. Organ cultures were incubated in media with and without added factors such as Dex or retinoic acid (RA) for 7 days in a 95% air-5% CO2 environment at 37°C. Media were changed daily under sterile conditions. The lungs were divided into two treatment groups (10 nM Dex and 10Morphology
The time 0 GD14 fetal mouse lungs and those cultured for 7 days as untreated controls or in the presence of 10 mM Dex or 10Total RNA Extraction
Total RNA was extracted from cultured untreated control and Dex-treated lungs with the original acid guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (7) or with its modified single-step, improved version (Ultraspec-II RNA Isolation Kit, Biotecx Laboratories, Houston, TX). After isolation and purification, the RNA was quantified at 260/280 nm with an ultraviolet (UV) spectrophotometer and stored atDifferential Display Procedure
Differential display was carried out with the protocols in the RNAimage Kit (GenHunter, Nashville, TN) as described by Liang and Pardee (17) and previously published by our laboratory (4, 28). All samples were run in duplicate. The experiments were performed twice, and the differential display of mRNAs was repeated with a second set of pooled lungs.Reverse transcription of RNA. Reverse transcription (RT) of the purified total RNA from each experimental condition was performed. RT mixtures included distilled water (dH2O), RT buffer, 20 µM deoxynucleotide triphosphate (dNTP), 0.2 µg of RNA, and 0.2 µM anchor primer H-T11G. Reactions ran as follows: 5 min at 65°C, 60 min at 37°C, 5 min at 75°C, and finally at 4°C. After 10 min at 37°C, 200 U of Moloney murine leukemia virus reverse transcriptase were added to each tube, and the incubation was continued for 60 min. Anchor primer G was randomly chosen for these studies out of the three available primers, A, C, and G.
Polymerase chain reaction.
Polymerase chain reaction (PCR) of the RT mixtures was then carried out
on each sample with the anchor primer G and one of 24 arbitrary primers
as provided in the RNAimage kit. PCR mixtures included
dH2O, PCR buffer, 2 µM dNTP, 0.2 µM H-T11G,
2.5 µCi of [-33P]dATP (2,000 Ci/mmol), 1 U of
AmpliTaq DNA polymerase, 0.2 µM arbitrary primer, and the
above RT mixtures. PCR was performed at the following settings on a
thermocycler: 30 s at 94°C, 2 min at 40°C, 30 s at 72°C for 40 cycles, then 10 min at 72°C, and finally at 4°C. PCR products were
loaded onto a 6% denaturing DNA-sequencing gel and electrophoresed for
3.5 h at 60-W constant power with maximum 1,700 V. Each gel was
blotted onto Whatman 3M paper, covered with Saran wrap, and
vacuum-dried at 80°C for 2 h. Dried gel was then exposed to
X-ray film, and after 48 h, the autoradiogram was developed for
each gel. Autoradiograms were checked for differentially expressed cDNA bands.
Reamplification of cDNA.
Several cDNA bands showed differences in signal intensity between
control and Dex, indicating induction, inhibition, enhancement, or
reduction of expression by Dex compared with control. Differentially expressed cDNA bands of interest were cut from the dried gel and eluted
in 100 µl of dH2O. Each cDNA was precipitated in sodium acetate, 100% ethanol, and glycogen overnight at 80°C. Each
precipitated cDNA was then centrifuged, and each cDNA pellet was washed
with 85% ethanol and then dissolved in 10 µl of dH2O.
Next, each cDNA was reamplified with the same primer set that was used
to obtain the respective bands. However, the dNTP concentration was
changed to 20 µM, and no isotope was added. PCR settings were
identical to those described in Polymerase chain
reaction. Reamplified cDNAs were run on 2% agarose gels
stained with ethidium bromide. Reamplified cDNA was extracted from the
agarose gel with a QIAEX Kit (QIAGEN, Chatsworth, CA).
cDNA Cloning
PCR-amplified cDNA fragments were cloned with the protocols in the PCR-TRAP cloning system version 2.0 (GenHunter).Ligation.
cDNA fragments were ligated into the PCR-TRAP vector overnight (21
h) at 16°C. Ligation reactions included dH2O, 300 ng of PCR-TRAP cloning vector, ligation buffer, PCR product, and 200 U/µl
of T4 DNA ligase.
Transformation and plating the cells. These protocols were based on the GenHunter PCR-TRAP cloning system protocol. Ligation mixture (10 µl) was added to 100 µl of GH-competent cells and incubated on ice for 45 min. Cells were heat shocked for 2 min at 42°C and then placed on ice. To these tubes, 400 µl of Luria Bertani (LB) broth was added, then they were incubated for 60 min at 37°C. Two hundred microliters of cells were plated onto LB plates containing 10 µg/ml of tetracycline. These were incubated overnight at 37°C, and the tetracycline-resistant colonies were scored the next morning as previously described (4).
Plasmid purification, denaturation, and sequencing the cloned PCR
products.
Each colony was picked with a pipette tip and injected into 5 ml of LB
culture broth containing 10 µg/ml of tetracycline. Cultures were
grown overnight at 37°C in a shaking incubator. Plasmid DNA (pDNA)
was purified from the cultures with the protocol in the
WizardPlus minipreps DNA purification system (Promega, Madison, WI) (4). Purified pDNA was denatured with 2 M
sodium hydroxide and 2 mM EDTA for 30 min at 37°C. It was then
precipitated with 3 M sodium acetate and 100% ethanol overnight at
80°C. pDNA was pelleted and dissolved in 7 µl of
dH2O.
Verification of cDNA. Analyses of each of the reamplified cDNAs were done on 2% agarose gels. The ethidium bromide-stained cDNA bands were visualized on an UV illuminator, cut out of the gel, and transferred to appropriately labeled microfuge tubes. Each cDNA was extracted from the gel with the QIAEX extraction kit (QIAGEN).
Northern Blot Analysis
We evaluated the dose response of Dex and RA and the time course for the effect of 10 nM Dex on lungs in culture after 7 days with Northern blot analysis. We extracted total RNA from control, Dex-treated, RA-treated, and normally developing lungs at various gestational stages and electrophoresed them on 1% agarose gels containing formaldehyde (1). Size-separated RNA was capillary transferred onto Gene Screen nylon membranes with 20× saline-sodium citrate (SSC; 1× SSC = 150 mM NaCl and 15 mM sodium citrate). After an overnight transfer, the RNA was UV cross-linked to the membrane. [32P]dCTP (3,000 Ci/mmol) was used to generate the respective cDNA probes by random priming with Klenow enzyme. Membranes were prehybridized in a solution consisting of 5× SSC, 5× Denhardt's solution (1), 50% formamide, 1% SDS, and 10 mg/ml of denatured salmon sperm DNA at 42°C for at least 4 h. Overnight hybridization was performed with each probe (5 × 105 counts · minProtein Determination
A minimum of three to five murine fetal lungs were pooled for preparing each sample. The lungs were homogenized on ice in PBS (pH 7.5) with protease inhibitors (1 µg/ml of aprotinin, 2 µg/ml of antipain, and 2 µg/ml of leupeptin). Total protein concentrations were analyzed by taking aliquots for the Bio-Rad microassay for protein (Bio-Rad, Hercules, CA) with the Bradford method (3) as previously described (2, 5). We also compared uncultured lungs from different developmental stages with the lungs cultured in the presence and absence of added factors such as Dex and RA.Immunoblot Analyses
The immunoblot analysis of the lungs was carried out as previously described (6). In this study, a mouse monoclonal antibody for p21ras protein was used as a primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). This was done to indirectly verify our results on the rrg from differential display of mRNAs and to prove our hypothesis that because rrg was enhanced by Dex, it would reduce the p21ras protein.Statistical Analyses
For differential display of mRNAs, samples were run in duplicate, and the experiments were repeated twice to confirm the altered expression of mRNAs before the cDNAs were isolated and sequenced. Semiquantitative analyses of the immunoblots were done by calculating the means ± SE. ANOVA was used to compare the data among the lungs cultured in the presence and absence of Dex as well as those cultured in the presence of other growth- and differentiation-promoting agents. When a difference was found, Dunnett's procedure for multiple comparisons was applied, with P set at 0.05 (20). ![]() |
RESULTS |
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Morphology
The gross morphology of cultured GD14 (time 0) lungs and of lungs cultured for 7 days was compared under a dissecting stereomicroscope. The time 0 lungs were pseudoglandular lungs. After 7 days in culture, the untreated control lungs showed little airway development. At the doses used, the lungs treated with Dex revealed prominent airways compared with the untreated lungs, with little evidence of saccular or alveolar development. In contrast, the lungs treated with RA for 7 days had significant saccular development as noted during the gross morphological observations. The sizes of the lungs under various conditions were measured with a dissecting seteromicroscope equipped with a metric ruler. The average length of the left lobe of time 0 GD14 fetal murine lungs was 2.0 ± 0.2 mm (n = 12). The RA-treated lungs (2.2 ± 0.2 mm) were of similar size to time 0 lungs, whereas the Dex-treated lungs were almost twice that size (4.0 ± 0.3 mm; P < 0.05; n = 12).Histologically, the coronal sections of time 0 lungs had the
characteristic features of the pseudoglandular lungs (Fig.
1A). After 7 days in culture,
the untreated control lungs had poorly defined airway branching and
little acinar development (Fig. 1B). The lungs that were
cultured in the presence of Dex had well-defined airways, thick acinar
walls, and an absence of secondary septation; however, surfactant
secretion was seen in the acinar spaces (Fig. 1C). The lungs
cultured in the presence of RA showed formation of saccular structures
and significant thinning of the saccular walls. Unlike control and
Dex-treated lungs, the RA-treated lungs also showed the formation of
secondary septa.
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Differentially Expressed Genes
The summary of the differentially expressed cDNAs in the murine fetal lungs cultured in the presence of Dex is given in Table 1. Anchor primer G, in combination with one of the 24 arbitrary primers used, yielded a total of 35 differentially expressed cDNAs in the Dex-treated lungs compared with that in the untreated control lungs. Of these differentially expressed cDNAs, 13 were enhanced by Dex, 14 were reduced, 1 was induced, and 7 were inhibited compared with the control lungs (Table 1). A representative autoradiograph demonstrating the differentially expressed cDNAs is shown in Fig. 2.
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On sequencing these differentially expressed cDNAs and identifying them
through the BLAST searches, we found that of the 13 cDNAs that were
enhanced by Dex, 3 specific cDNAs (which we named Dex1, Dex2, and Dex4)
were identified as rrg or LOX gene (Table 2). Dex2 had 100% sequence similarity
with the existing LOX gene in the GenBank database (locus MUSLYSOX,
accession number M65142), and Dex1 and Dex4 had 92-97% similarity
with the LOX gene. Furthermore, the cDNA termed Dex3, which was induced
by Dex, did not have any similarity with the existing gene sequences in
the database. Therefore, we labeled it as an unknown gene (Table
2). Another cDNA, termed UN1, which had a greater expression in
untreated control lungs and was reduced by Dex in the cultured lungs,
showed 98% sequence similarity with the fibroblast growth factor
receptor 3 (FGFR3) gene (Table 2). Out of the cDNAs sequenced so far,
~25% have been false positive, and some others have been identified
as the known structural genes, with low probability ratios. Several
other cDNAs genes are currently being sequenced.
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A Northern blot confirmation of rrg or LOX gene
was carried out in normally developing lungs ranging in age from GD14
to neonates and also in in vitro control lungs. It was also carried out
in the lungs treated with Dex and RA (Fig.
3). The results clearly revealed
upregulation of rrg by Dex. RA lungs demonstrated
rrg expression similar to that in neonatal lungs, but this
expression was significantly lower than that seen in the presence of
Dex (Fig. 3) or even in the untreated control lungs.
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Immunoblot Analyses
We carried out the Western blot analyses for p21ras protein based on our hypothesis that because the rrg is enhanced by Dex, therefore p21ras protein must be suppressed by Dex. Our immunoblot results revealed very little expression of p21 in developing lungs in vivo from GD14 to neonatal stage. Furthermore, all cultured lungs in the presence or absence of added factors showed higher levels of p21 (the Ras protein) compared with lungs developing in vivo. Our results showed that p21ras gene product (protein) was downregulated by Dex in the lungs (Fig. 4). This effect was complementary to the upregulation of rrg by Dex observed in our study of the differential display of mRNAs.
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Others (13) have shown that in adipose tissue, RA reduced
the expression of rrg, and our current study revealed that
Dex enhances the expression of rrg. Upregulation of the
rrg, as the name suggests, is known to downregulate the
expression of p21ras protein. Because RA is
known to reduce rrg expression, it may upregulate
p21ras protein. To confirm our current results,
we compared p21ras protein levels in lungs
treated with Dex with those treated with RA. At the dose of RA used in
this study (105 M), we observed increased levels of
p21ras protein in fetal lungs in culture (Fig.
4). This observation suggested opposite effects of Dex and RA as well
as indirectly corroborated our results from the differential display of
mRNAs. In the lungs cultured in the presence of a combination of Dex and RA, the level of p21ras protein was
decreased (Fig. 4), indicating the predominant effects of Dex on this protein.
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DISCUSSION |
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We observed that Dex upregulated the rrg, also known as
the LOX gene. LOX is a copper-dependent extracellular
metalloenzyme that plays a central role in the cross-linking of
collagen and elastin in the extracellular matrix (ECM) (i.e.,
maturation of collagen and elastin, as reviewed in Ref. 23). LOX
catalyzes the oxidation of lysine residues to -aminoadipic
-semialdehyde. This is the first step in the covalent cross-linking
of collagen and tropoelastin, and it results in the formation of
insoluble collagen and elastin fibers in the ECM. LOX gene
or rrg activity has been subsequently found to be increased
in fibrotic lung disease (15).
Because rrg plays a role in collagen-elastin cross-linking, we speculate that, in our study, upregulation of rrg by Dex resulted in formation of collagen and elastin fibers in ECM. Furthermore, these results corroborated our morphological observations, which demonstrated that there were thick septa present in these lungs, and that although the air spaces were larger, no secondary septa developed in Dex-treated lungs. One of the ECM components, elastin, is pivotal to the recoil of the lung vasculature and the alveolar walls. Abnormal elastin metabolism is associated with several diseases of the lung, including pulmonary hypertension (19) and emphysema (12). In the absence of any pulmonary disease, the normal adult lung produces little elastin (24); therefore, lung function during one's lifetime depends on the elastin produced during development.
It has been shown that inhibitors of LOX administered during development result in abnormal air space morphology (11, 16). This disruption leads to persistent abnormalities in alveolar size and number. Similar persistent effects have been reported in rats maintained on a copper-free diet, which reduced LOX activity (22). These findings are consistent with a critical role for cross-linked elastin or collagen in fetal lung development. Increased activity of LOX may result in lung fibrosis, and inhibition of it may lead to abnormal air space morphology. Therefore, we suggest that maintaining the balance of the activity of LOX is crucial to the normal development of the lung.
cDNA to the human LOX has been isolated, and the gene has been mapped to human chromosome 5q23.3-q31.2 (14, 18). It is a single gene in which the locus is distal to two tumor suppressor genes. Its structure has been partially characterized (25). The mouse LOX gene has been mapped to mouse chromosome 18, distal to Camk-4 and proximal to li, extending a region that is syntenic with human chromosome 5q (21). LOX gene is a single copy gene organized into seven exons and six introns, and it spans ~14 kb of the mouse genome (10). The gene encodes two messages, sized at ~4.8 and 3.8 kb, that differ in length of the untranslated sequence at the 3'-end of the gene. All of the 3'-untranslated sequence and the polyadenylation signals are contained in exon 7; there is no evidence of alternate splicing (10).
Through differential localization of Ras proteins and variations in their levels, it has been proposed that Ras proteins may be important during lung growth and differentiation. In fetal rat lungs, proliferative activity is known to decline near birth, followed by a gradual increase in cellular proliferation during the subsequent 8 days and a decline in basal levels by 15-18 days after birth (26). During this period of substantial variation in proliferative activity, differences in both the protein content and localization of the different Ras proteins have been observed.
Interestingly, the enzyme LOX (an rrg product) also appears to function as a phenotypic suppressor of the ras oncogene product p21 in NIH/3T3 cells (10). It is known that ras-transformed oncogenic cell detachment might be due to cytoskeletal and/or cell matrix alterations. Furthermore, filamentous intracellular localization of LOX in fibroblasts, smooth muscle cells, and some cloned cell lines demonstrated that it is associated with cytoskeletal protein (27). These cytoskeletal elements involve microtubules, actin, and other microfilaments and intermediate filaments. The alterations in the cytoskeletal elements result in tissue-specific changes in cell shape, movement, and interactions with growth factors. Taken together, it is suggested that the ECM, along with the cytoskeleton and nuclear matrix, may regulate tissue-specific gene expression.
Furthermore, it has been known that nontumorigenic reverants transfected with rrg (LOX gene) antisense constructs became retransformed and tumorigenic, and rrg message levels dropped to prereversion levels. It has been demonstrated that LOX or the rrg product has tumor suppressor activity and that it also plays a role in several pathological conditions. This indicates that the state of the connective tissue matrix, as influenced by LOX, is important in the prevention of the uncontrolled growth of oncogenic cells as well as in normal cell function.
We conducted a comparative study with and without added factors to the
fetal lungs in culture and assessed the levels of
p21ras protein in these lungs. Because Dex
upregulated rrg activity (a suppressor of
p21ras protein) in our differential display
analyses, we expected to see downregulation of
p21ras protein in Dex-treated lungs. Our
immunoblot data were supportive of this hypothesis. Furthermore, our
laboratory (9) has previously shown that 10 nM Dex and
105 M RA anatagonized several biochemical and
morphological features in the developing lung. A comparison of
p21ras protein in lungs cultured in the presence
of RA or Dex revealed that RA, unlike Dex, upregulated this protein. It
was also noted that the p21 level was enhanced in vitro under all
conditions. Addition of Dex or RA had specific influence on the levels
of p21 protein. Functional assays assessing specific roles of LOX and
p21ras protein in the developing lungs will shed
light on specific aspects of the regulation of normal lung development.
In summary, Dex treatment results in differential gene expression compared with that in control murine fetal lungs in culture. Based on our results and on existing literature, we speculate the following. 1) The regulation of LOX via Dex-enhanced expression affects the connective tissue matrix in the developing murine lung, which is indirectly supported by the presence of thick septa in these lungs. 2) Enhanced expression of rrg may be correlated to the increased thickness of the acinar septa and inhibition of secondary septa formation in the Dex-treated lungs. 3) It is plausible that in addition to accelerating maturation and differentiation of the developing lung, Dex may also adversely alter the connective tissue matrix in such a way that saccularization or alveolarization is inhibited, or it may contribute to fibrotic lung formation. Therefore, a balance in the ECM is crucial for promoting normal lung development. 4) From a therapeutic point of view, these observations raise a further question. Does continued influence by Dex on the developing lung in vitro or in vivo adversely affect the ECM (e.g., resulting in fibrosis or inhibition of alveolar formation) in addition to its desired effect of accelerating lung epithelial cell maturation and differentiation?
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ACKNOWLEDGEMENTS |
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We thank Dr. Li Zhang for assistance in the differential display of mRNAs, Dr. Xiaoli Chi for her assistance in the Western blot analyses, and Roland L. Myers and Lisa McCully for assistance in preparation of the figures.
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FOOTNOTES |
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This research was supported in part by Clinical Research Grant 6-FY97-0147 and Basic Research Grant 6-FY98-0608 from the March of Dimes Birth Defects Foundation, and an American Lung Association Career Investigator Award (M. R. Chinoy).
Address for reprint requests and other correspondence: M. R. Chinoy, Lung Development Research Program, Dept. of Surgery, H 113, Pennsylvania State Univ., Milton S. Hershey Medical Center, Hershey, PA 17033 (E-mail: mchinoy{at}psu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 9 July 1999; accepted in final form 21 March 2000.
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