Sustained changes in lung expansion alter tropoelastin mRNA levels and elastin content in fetal sheep lungs

2003 ◽  
Vol 284 (4) ◽  
pp. L643-L649 ◽  
Author(s):  
Belinda J. Joyce ◽  
Megan J. Wallace ◽  
Richard A. Pierce ◽  
Richard Harding ◽  
Stuart B. Hooper
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Fetal Lung ◽  
Elastic Fibers ◽  
Mrna Levels ◽  
Fetal Sheep ◽  
Time Points ◽  
Tracheal Obstruction ◽  
Lung Expansion

Our objective was to determine the effects of sustained alterations in fetal lung expansion on pulmonary elastin synthesis. In fetal sheep, lung expansion was either decreased between 111 and 131 days' gestation (term ∼147 days) by tracheal drainage or increased for 2, 4, 7, or 10 days by tracheal obstruction, ending at 128 days' gestation. Lung tropoelastin mRNA levels were assessed by Northern blot analysis, total elastin content was measured biochemically, and staining of lung sections was used to assess the localization and form of elastic fibers. Tracheal obstruction significantly elevated pulmonary tropoelastin mRNA levels 2.5-fold at 2 days, but values were not different from controls at 4, 7, and 10 days; elastin content tended to be increased at all time points. A sustained decrease in lung expansion by tracheal drainage reduced pulmonary tropoelastin mRNA levels 2.5-fold; elastin content was also decreased compared with controls, and tissue localization was altered. Our results indicate that the degree of lung expansion in the fetus influences elastin synthesis, content, and tissue deposition.

Alterations in lung expansion affect surfactant protein A, B, and C mRNA levels in fetal sheep

1999 ◽  
Vol 276 (2) ◽  
pp. L239-L245 ◽  
Author(s):  
A. Lines ◽  
L. Nardo ◽  
I. D. Phillips ◽  
F. Possmayer ◽  
S. B. Hooper
Keyword(s):  
Lung Tissue ◽  
Blot Analysis ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Mrna Levels ◽  
Lung Growth ◽  
Fetal Sheep ◽  
Protein Levels ◽  
Tracheal Obstruction ◽  
Lung Expansion

Obstruction of the fetal trachea is a potent stimulus for fetal lung growth, and it has been suggested that this procedure may be used therapeutically to reverse lung growth deficits in human fetuses with lung hypoplasia. However, little is known about the effects of increased lung expansion on other aspects of lung development. Our aim was to determine the effect of increased and decreased lung expansion on the mRNA levels encoding surfactant protein (SP) A, SP-B, and SP-C in ovine fetal lungs. Lung tissue samples were collected from fetuses exposed to 2, 4, or 10 days of increased lung expansion caused by tracheal obstruction. The mRNA levels for SP-A, SP-B, and SP-C were determined by Northern blot analysis with specific ovine cDNA probes; SP-A protein levels were determined by Western blot analysis. Compared with age-matched (128-day gestational age) control fetuses, SP-A, SP-B, and SP-C mRNA levels in fetal lung tissue were significantly reduced at 2 days of tracheal obstruction and remained reduced at 4 and 10 days. However, SP-A protein levels were not reduced at 2 days of tracheal obstruction, tended to be reduced at 4 days, and were almost undetectable at 10 days. In contrast to tracheal obstruction, 7 days of lung liquid drainage significantly increased SP-C, but not SP-A, mRNA levels in fetal lung tissue compared with age-matched control fetuses. Our results demonstrate that increases in fetal lung expansion, induced by obstruction of the fetal trachea, cause large simultaneous reductions in SP-A, SP-B, and SP-C mRNA levels in the fetal lung as well as a decrease in SP-A protein levels. These data suggest that expression of the genes encoding SPs in the fetal lung are specifically responsive to the degree of lung expansion.

Changes in lung expansion alter pulmonary DNA synthesis and IGF-II gene expression in fetal sheep

1993 ◽  
Vol 265 (4) ◽  
pp. L403-L409 ◽  
Author(s):  
S. B. Hooper ◽  
V. K. Han ◽  
R. Harding
Keyword(s):  
Gene Expression ◽  
Dna Synthesis ◽  
Fetal Lung ◽  
Liquid Volume ◽  
Mrna Levels ◽  
Pulmonary Tissue ◽  
Fetal Sheep ◽  
Tracheal Obstruction ◽  
Lung Expansion ◽  
Lung Liquid

Our aim was to determine the effect of short-term (7 days) alterations in fetal lung liquid volume on pulmonary DNA synthesis rates and insulin-like growth factor-II (IGF-II) mRNA levels. Fifteen chronically catheterized fetal sheep were divided into three groups. In one, the trachea was obstructed, in another lung liquid was drained by gravity, and the third group served as controls. After 7 days, [3H]thymidine was injected into each fetus and 8 h later fetal tissues were collected. Fetal lung-to-body weight ratios and total lung DNA contents were greatly increased in fetuses with tracheal obstruction compared with control fetuses, whereas the drainage of lung liquid did not affect these measurements. DNA synthesis rates in pulmonary tissue were significantly reduced from a mean control value of 153.3 +/- 25.1 disintegrations per minute (dpm)/microgram DNA to 57.2 +/- 8.6 dpm/microgram DNA by lung liquid drainage (P < 0.05) and were significantly increased to 236.0 +/- 24.0 dpm/microgram DNA by tracheal obstruction (P < 0.05). Following tracheal obstruction, lung IGF-II mRNA levels were increased to 177.0 +/- 18.2% (P < 0.05) of the mean value for control fetuses, whereas they were reduced to 56.1 +/- 7.1% of control in lung liquid-drained fetuses. We conclude that altering fetal lung expansion has a potent and rapid effect on pulmonary DNA synthesis and that this effect may, in part, be mediated by an alteration in IGF-II gene expression.

scholarly journals Increased expansion of the lung stimulates calmodulin 2 expression in fetal sheep

2002 ◽  
Vol 282 (3) ◽  
pp. L440-L447 ◽  
Author(s):  
A. M. Gillett ◽  
M. J. Wallace ◽  
M. T. Gillespie ◽  
S. B. Hooper
Keyword(s):  
Northern Blot Analysis ◽  
Fetal Lung ◽  
Mrna Levels ◽  
Lung Growth ◽  
Coding Region ◽  
Fetal Sheep ◽  
Protein Levels ◽  
Lung Expansion ◽  
Rapid Amplification

Obstruction of the fetal trachea causes the lungs to expand with accumulated liquid. Although this is a potent stimulus for lung growth, the mechanisms involved are unknown. Our aim was to identify genes that are differentially expressed as a result of increased fetal lung expansion. Using differential display RT-PCR, we isolated a cDNA fragment partially encoding calmodulin 2 (CALM2) and identified the remainder of the coding region by 5′-rapid amplification of cDNA ends. Differential expression of CALM2 was confirmed by Northern blot analysis; CALM2 mRNA levels were increased to 161 ± 5% of control at 2 days of increased lung expansion, induced by tracheal obstruction (TO), and had returned to control levels at days 4 and 10. Using in situ hybridization analysis, we found that the proportion of CALM2-labeled cells increased from 10.3 ± 1.0% to 21.4 ± 6.8% by 2 days of TO. This increase in CALM2 expression was reflected by a tendency for calmodulin protein levels to increase from 122.7 ± 17.3 to 156.5 ± 17.7 at 2 days of TO. Thus increases in fetal lung expansion result in time-dependent changes in CALM2 mRNA levels, which closely parallels the changes in lung DNA synthesis rates. As calmodulin is essential for cell proliferation, increased CALM2 mRNA levels may reflect an important role for calmodulin in expansion-induced fetal lung growth.

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene

2004 ◽  
Vol 16 (8) ◽  
pp. 763 ◽  
Author(s):  
Han-Seung Kang ◽  
Chae-Kwan Lee ◽  
Ju-Ran Kim ◽  
Seong-Jin Yu ◽  
Sung-Goo Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Oestrous Cycle ◽  
Mrna Levels ◽  
Rat Uterus ◽  
Ovx Rats

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P4)-injected OVX rats (OVX v. OVX + P4 pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P4 treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [α32P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P4 pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P4 animals. The genes for nuclear factor I–XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin β-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P4 rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P4. One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17β-oestradiol (E2) and P4, although P4 administration upregulated gene expression to a greater extent than injection of E2. These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E2 and P4, which are secreted periodically during the oestrous cycle.

Prognostic Significance of the Combined Expression of Matrix Metalloproteinase-9, Urokinase Type Plasminogen Activator and its Receptor in Breast Cancer as Measured by Northern Blot Analysis

2001 ◽  
Vol 16 (1) ◽  
pp. 62-68 ◽  
Author(s):  
M.M. Pacheco ◽  
I.N. Nishimoto ◽  
M. Mourão Neto ◽  
E.B. Mantovani ◽  
M.M. Brentani
Keyword(s):  
Breast Cancer ◽  
Overall Survival ◽  
Plasminogen Activator ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Prognostic Significance ◽  
Mrna Levels ◽  
Upa Mrna ◽  
The Impact

Using Northern blot analysis we have measured the co-expression of the matrix metalloprotease MMP-9, plasminogen activator urokinase type (uPA) and its receptor (uPAR) mRNAs in 81 biopsies of breast carcinomas with the objective of analyzing the impact of these factors on the overall survival probability of the patients (median follow-up time: 4 years). Individual mRNA levels of either uPA or uPAR showed parallel variations with MMP-9 mRNA, suggesting a coordinate transcription of these markers. When median values were used as cutoff points to discriminate between high and low factor expression, no association was found with patient outcome and MMP-9 or uPA mRNA distribution. However, increased uPAR mRNA levels were associated with poor prognosis (p = 0.01). The combination of MMP-9 and uPAR mRNA measurements has not enhanced prognostic information compared to information supplied by the receptor alone (p = 0.01). The combination of MMP-9 and high levels of uPA mRNA led to a significant association with poor outcome (p = 0.03). Multivariate analysis supported the notion that increased uPAR mRNA production in primary breast cancer may be a predictor of overall survival.

scholarly journals Influence of growth hormone on the lung growth response to tracheal obstruction in fetal sheep

2000 ◽  
Vol 278 (3) ◽  
pp. L453-L459 ◽  
Author(s):  
L. Nardo ◽  
I. R. Young ◽  
S. B. Hooper
Keyword(s):  
Growth Hormone ◽  
Dna Content ◽  
Fetal Lung ◽  
Control Group ◽  
Lung Growth ◽  
Lung Weight ◽  
Fetal Sheep ◽  
Tracheal Obstruction ◽  
Lung Expansion ◽  
Lung Liquid

Obstructing the fetal trachea is a potent stimulus for fetal lung growth, but little is known about the factors that regulate this process. Our aim was to determine the role of growth hormone (GH) in regulating the increase in lung growth induced by obstruction of the trachea in fetal sheep. Twenty chronically catheterized fetal sheep, nine of which were hypophysectomized, were divided into four experimental groups: 1) control group ( n = 4), 2) a group in which the fetal trachea was obstructed for 3 days (3-day obstructed; n = 6), 3) a 3-day obstructed group in which the pituitary was removed [hypophysectomized (HX)] and the fetus was given maintenance infusions of ACTH, thyroxine, and human GH (hGH; HX hGH 3-day obstructed; n = 5), and 4) a HX 3-day obstructed group in which the fetus was given maintenance infusions of ACTH and thyroxine ( n = 5). Tracheal obstruction significantly increased fetal lung liquid volumes from 37.2 ± 3.2 ml/kg in control fetuses to 75.6 ± 9.0 ml/kg in 3-day obstructed fetuses, and the presence or absence of GH did not affect this increase. Similarly, the presence or absence of GH did not affect the increase in lung weight or protein content induced by 3 days of tracheal obstruction. However, in the absence of GH, 3 days of tracheal obstruction failed to increase total lung DNA content above unobstructed control values (107.9 ± 5.3 and 94.1 ± 7.0 mg/kg for control and HX 3-day obstructed groups, respectively). In contrast, 3 days of tracheal obstruction increased total lung DNA content to a similar extent in fetuses with an intact pituitary and HX fetuses that received GH replacement (126.0 ± 4.4 and 126.7 ± 4.0 mg/kg for 3-day obstructed and HX hGH 3-day obstructed groups, respectively). These data indicate that the absence of GH either abolishes or delays the acceleration in cell division caused by an increase in fetal lung expansion.

scholarly journals Three mRNA transcripts of the proopiomelanocortin gene in human placenta at term

2000 ◽  
pp. 533-536 ◽  
Author(s):  
SI Grigorakis ◽  
E Anastasiou ◽  
K Dai ◽  
A Souvatzoglou ◽  
M Alevizaki
Keyword(s):  
Pregnant Women ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Mode Of Delivery ◽  
Mrna Levels ◽  
Functional Protein ◽  
Mrna Transcripts ◽  
Gene Transcripts ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene whose normal pituitary specific mRNA product is 1200 bases (b) is also expressed in placenta and its peptide derivatives such as ACTH and beta-endorphin may play an important role in the initiation of labor. So far, two mRNA transcripts, one small (800b) and one large (1380b) have been reported in placenta by Northern blot analysis, similar to other endocrine tissues and various extrapituitary tumors; however, it is questionable whether both of these transcripts are effectively translated to a functional protein. We examined by Northern blot analysis the size and the differential expression of placental POMC gene transcripts in pregnant women with different modes of delivery. Placental tissues were collected from two groups of pregnant women, six with vaginal delivery (VD) and five with cesarean section (CS). In both groups of placentae three POMC gene transcripts were detected of 800, 1200 and 1380 bases; the 1200b pituitary specific species often predominated and was always present. The 800b transcript was also always present, while the large transcript (1380b) was expressed in 3/6 VD and 2/5 CS placental tissues. No differences in the relative levels of any of these mRNA species showing effect of the mode of delivery were observed. We conclude that POMC gene transcription in placental tissue at term gives rise to three mRNA transcripts, thus resembling extrapituitary tumors. The reported changes in the levels of the derivative peptides according to the mode of delivery do not reflect changes in POMC mRNA levels and could be attributed to a post-translational effect.

scholarly journals Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization.

1989 ◽  
Vol 108 (5) ◽  
pp. 1823-1832 ◽  
Author(s):  
O Horovitz ◽  
D Knaack ◽  
T R Podleski ◽  
M M Salpeter
Keyword(s):  
Ascorbic Acid ◽  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Alpha Subunit ◽  
Mrna Levels ◽  
Surface Receptors ◽  
Subunit Mrna

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.

Regulation of alveolar epithelial cell phenotypes in fetal sheep: roles of cortisol and lung expansion

2004 ◽  
Vol 287 (6) ◽  
pp. L1207-L1214 ◽  
Author(s):  
Sharon J. Flecknoe ◽  
Rochelle E. Boland ◽  
Megan J. Wallace ◽  
Richard Harding ◽  
Stuart B. Hooper
Keyword(s):  
Epithelial Cell ◽  
Alveolar Epithelial Cell ◽  
Fetal Lung ◽  
Saline Infusion ◽  
Type I ◽  
Mrna Levels ◽  
Type Ii ◽  
Fetal Sheep ◽  
Alveolar Epithelial ◽  
Lung Expansion

Our aim was to determine whether cortisol's effect on alveolar epithelial cell (AEC) phenotypes in the fetus is mediated via a sustained alteration in lung expansion. Chronically catheterized fetal sheep were exposed to 1) saline infusion, 2) cortisol infusion (122–131 days' gestation, 1.5–4.0 mg/day), 3) saline infusion plus reduced lung expansion, or 4) cortisol infusion plus reduced lung expansion. The proportions of type I and II AECs were determined by electron microscopy, and surfactant protein (SP)-A, -B, and -C mRNA levels were determined by Northern blot analysis. Cortisol infusions significantly increased type II AEC proportions (to 38.2 ± 2.2%), compared with saline-infused fetuses (23.8 ± 2.4%), and reduced type I AEC proportions (to 59.0 ± 2.2%), compared with saline-infused fetuses (70.4 ± 2.4%). Reduced lung expansion also increased type II AEC proportions (to 52.9 ± 3.5%) and decreased type I AEC proportions (to 34.2 ± 3.7%), compared with control, saline-infused fetuses. The infusion of cortisol into fetuses exposed to reduced lung expansion tended to further increase type II (to 60.3 ± 2.1%, P = 0.066) and reduce type I AEC (to 26.6 ± 2.3%, P = 0.07) proportions. SP-A, -B, and -C mRNA levels changed in parallel with the changes in type II AEC proportions. These results indicate that cortisol alters the proportion of type I and type II AECs via a mechanism unrelated to the degree of fetal lung expansion. However, reductions in fetal lung expansion appear to have a greater impact on the proportion of AECs than cortisol.

Distribution and developmental pattern of neuromedin U expression in the rat gastrointestinal tract

1994 ◽  
Vol 12 (3) ◽  
pp. 257-263 ◽  
Author(s):  
C Austin ◽  
M Oka ◽  
K A Nandha ◽  
S Legon ◽  
N Khandan-Nia ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Postnatal Development ◽  
Food Deprivation ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Developmental Pattern ◽  
Mrna Levels ◽  
First Time

ABSTRACT This study has quantified, for the first time, the relative levels of neuromedin U (NmU) mRNA in the rat gastrointestinal tract using Northern blot analysis. NmU message was detected in all regions of the gastrointestinal tract from the oesophagus to the rectum. The greatest levels were found in the duodenum and jejunum, the principal sites for absorption, which were 2·5- and 3-fold respectively above ileal levels. Quantification of NmU mRNA and peptide contents in the duodenum, jejunum and ileum during postnatal development of the rat showed message and peptide levels to be greater in the maturing rat than in neonates. Message levels in the duodenum, jejunum and ileum showed 14-, 7- and 4-fold increases respectively between 1 and 56 days after birth, whilst the corresponding peptide levels in the duodenum, jejunum and ileum showed 33-, 14- and 25-fold increases respectively. Food deprivation caused a small, but significant, decrease in message levels in the jejunum and colon, but there was no change in the duodenum or ileum. This shows that the presence of food has little effect on NmU mRNA levels in the gut.

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