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1.
JCO Precis Oncol ; 20172017.
Article in English | MEDLINE | ID: mdl-30761385

ABSTRACT

PURPOSE: Genomic testing has increased the quantity of information available to oncologists. Unfortunately, many identified sequence alterations are variants of unknown significance (VUSs), which thus limit the clinician's ability to use these findings to inform treatment. We applied a combination of in silico prediction and molecular modeling tools and laboratory techniques to rapidly define actionable VUSs. MATERIALS AND METHODS: Exome sequencing was conducted on 308 tumors from various origins. Most single nucleotide alterations within gene coding regions were VUSs. These VUSs were filtered to identify a subset of therapeutically targetable genes that were predicted with in silico tools to be altered in function by their variant sequence. A subset of receptor tyrosine kinase VUSs was characterized by laboratory comparison of each VUS versus its wild-type counterpart in terms of expression and signaling activity. RESULTS: The study identified 4,327 point mutations of which 3,833 were VUSs. Filtering for mutations in genes that were therapeutically targetable and predicted to affect protein function reduced these to 522VUSs of interest, including a large number of kinases. Ten receptortyrosine kinase VUSs were selected to explore in the laboratory. Of these, seven were found to be functionally altered. Three VUSs (FGFR2 F276C, FGFR4 R78H, and KDR G539R) showed increased basal or ligand-stimulated ERK phosphorylation compared with their wild-type counterparts, which suggests that they support transformation. Treatment of a patient who carried FGFR2 F276C with an FGFR inhibitor resulted in significant and sustained tumor response with clinical benefit. CONCLUSION: The findings demonstrate the feasibility of rapid identification of the biologic relevance of somatic mutations, which thus advances clinicians' ability to make informed treatment decisions.

2.
J Vis Exp ; (78)2013 Aug 27.
Article in English | MEDLINE | ID: mdl-24022340

ABSTRACT

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.


Subject(s)
Epithelial-Mesenchymal Transition/physiology , Immunohistochemistry/methods , Cell Line, Tumor , Cell Movement/physiology , Humans
3.
BMC Biotechnol ; 9: 59, 2009 Jun 29.
Article in English | MEDLINE | ID: mdl-19563662

ABSTRACT

BACKGROUND: The investigation of molecular mechanisms underlying transcriptional regulation, particularly in embryonic stem cells, has received increasing attention and involves the systematic identification of target genes and the analysis of promoter co-occupancy. High-throughput approaches based on chromatin immunoprecipitation (ChIP) have been widely used for this purpose. However, these approaches remain time-consuming, expensive, labor-intensive, involve multiple steps, and require complex statistical analysis. Advances in this field will greatly benefit from the development and use of simple, fast, sensitive and straightforward ChIP assay and analysis methodologies. RESULTS: We initially developed a simplified, basic ChIP protocol that combines simplicity, speed and sensitivity. ChIP analysis by real-time PCR was compared to analysis by densitometry with the ImageJ software. This protocol allowed the rapid identification of known target genes for SOX2, NANOG, OCT3/4, SOX17, KLF4, RUNX2, OLIG2, SMAD2/3, BMI-1, and c-MYC in a human embryonic stem cell line. We then developed a novel Sequential ChIP protocol to investigate in vivo promoter co-occupancy, which is basically characterized by the absence of antibody-antigen disruption during the assay. It combines centrifugation of agarose beads and magnetic separation. Using this Sequential ChIP protocol we found that c-MYC associates with the SOX2/NANOG/OCT3/4 complex and identified a novel RUNX2/BMI-1/SMAD2/3 complex in BG01V cells. These two TF complexes associate with two distinct sets of target genes. The RUNX2/BMI-1/SMAD2/3 complex is associated predominantly with genes not expressed in undifferentiated BG01V cells, consistent with the reported role of those TFs as transcriptional repressors. CONCLUSION: These simplified basic ChIP and novel Sequential ChIP protocols were successfully tested with a variety of antibodies with human embryonic stem cells, generated a number of novel observations for future studies and might be useful for high-throughput ChIP-based assays.


Subject(s)
Chromatin Immunoprecipitation/methods , Embryonic Stem Cells/cytology , Animals , Cell Line , Embryonic Stem Cells/metabolism , Humans , Kruppel-Like Factor 4 , Mice , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Software
4.
Proc Natl Acad Sci U S A ; 104(47): 18789-94, 2007 Nov 20.
Article in English | MEDLINE | ID: mdl-18000055

ABSTRACT

The transcriptional machinery involved in the transition of an infant from intrauterine to air-breathing life is developmentally regulated, as the fetus and adult manifest differential genetic expression. The low oxygen (O(2)) environment of the mammalian fetus and the increase in O(2) tension that occurs at birth may account for the developmentally regulated alterations in gene expression. We tested the hypothesis that hypoxia-inducible factor 1 (HIF-1) expression, an O(2)-sensitive transcription factor, is developmentally regulated. We found that in fetal pulmonary artery (PA) smooth muscle cells (SMC), fetal HIF-1 protein levels were O(2)-insensitive, whereas in adult PA SMC, hypoxia increased HIF-1 protein expression. Surprisingly, hypoxia increased HIF-1 mRNA expression in fetal, but not in adult, PA SMC. HIF-1 degradation and transcriptional activity is contingent on prolyl- and asparagyl-hydroxylases. To determine whether developmental differences in O(2) sensitivity or expression of these enzymes accounts for the divergence of HIF-1 sensitivity between fetus and adult, we studied the expression of the three most well characterized prolyl-hydroxylases, PHD1, PHD2, and PHD3, and the expression of regulators of HIF-1 transcriptional activity, asparagyl-hydroxylase, factor inhibiting HIF, and the oncogenic factor, CITED2 (CREB-binding protein/p300 interacting transactivator with ED-rich tail). We found that, as in the case of HIF-1, these genes are differentially regulated in the fetus, enabling the mammalian fetus to thrive in the low O(2) tension intrauterine environment even while rendering a newborn infant uniquely well adapted to respond to the acute increase in O(2) tension that occurs at birth.


Subject(s)
Gene Expression Regulation, Developmental , Hypoxia-Inducible Factor 1/metabolism , Lung/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Procollagen-Proline Dioxygenase/metabolism , Aging/physiology , Animals , Cells, Cultured , Hypoxia-Inducible Factor 1/genetics , Procollagen-Proline Dioxygenase/genetics , RNA, Messenger/genetics , Repressor Proteins/metabolism , Sheep
5.
Gene Expr Patterns ; 7(1-2): 8-14, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16798105

ABSTRACT

Efforts to characterize the mechanisms underlying early lung development have been confounded by the absence of a model that permits study of lung development prior to the onset of endodermal differentiation. Since Xenopus laevis development occurs in an extrauterine environment, we sought to determine whether the classical molecular markers of lung development and function, surfactant protein genes, are expressed in X. laevis. Surfactant protein C (SP-C) is a specific marker for lung development, expressed early in development and exclusively in the lung. Surfactant protein B (SP-B) expression is essential for life, as its absence results in neonatal death in mice and gene mutations have been associated with neonatal respiratory failure in humans. Here, we report the cloning of the first non-mammalian SP-C and SP-B genes (termed xSP-C and xSP-B) using the Xenopus model. The processed mature translated regions of both xSP-C and xSP-B have high homology with both human and mouse genes. xSP-C and xSP-B are both expressed throughout the lung of the X. laevis swimming tadpoles soon after the initiation of lung development as assessed by RT-PCR and whole mount in situ hybridization. The temporal expression patterns of xSP-C and xSP-B are consistent with the expression patterns in mammalian models of lung development. In both the tadpole and the adult X. laevis, xSP-C and xSP-B are expressed only in lung. Knowledge of the sequence and expression pattern of these two surfactant proteins in Xenopus might allow for use of this organism to study early lung development.


Subject(s)
Lung/growth & development , Lung/metabolism , Pulmonary Surfactant-Associated Protein B/genetics , Pulmonary Surfactant-Associated Protein C/genetics , Xenopus laevis/growth & development , Xenopus laevis/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers/genetics , Gene Expression Regulation, Developmental , Humans , In Situ Hybridization , Infant, Newborn , Mice , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Tissue Distribution
6.
Am J Physiol Lung Cell Mol Physiol ; 292(4): L953-9, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17158601

ABSTRACT

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.


Subject(s)
Calcium/metabolism , Fetal Diseases/metabolism , Hypertension, Pulmonary/metabolism , Muscle, Smooth, Vascular/metabolism , Pulmonary Artery/metabolism , Animals , Blotting, Western , Calcium Channels/metabolism , Calcium Signaling/physiology , Disease Models, Animal , Female , Humans , Hypertension, Pulmonary/embryology , Infant, Newborn , Muscle, Smooth, Vascular/cytology , Persistent Fetal Circulation Syndrome/metabolism , Pregnancy , Pulmonary Artery/cytology , Sheep , Transient Receptor Potential Channels/physiology
7.
Pediatr Res ; 60(3): 258-63, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16857761

ABSTRACT

To test the hypothesis that an acute increase in O(2) tension increases cytosolic calcium ([Ca(2+)](i)) in fetal pulmonary artery endothelial cells (PAECs) via entry of extracellular calcium and subsequent calcium-induced calcium release (CICR) and nitric oxide release, low-passage PAECs (<10 passages) were isolated from the intralobar pulmonary artery (PA) of fetal sheep and maintained under hypoxic conditions (Po(2), 25 Torr). Using the calcium-sensitive dye fura-2, we demonstrated that acute normoxia (Po(2) = 120 Torr) increased PAECs [Ca(2+)](i) by increasing the rate of entry of extracellular calcium. In the presence of either ryanodine or 2-aminoethoxy-diphenylborate (2APB), normoxia did not lead to a sustained increase in PAECs [Ca(2+)](i) Whole-cell patch clamp studies demonstrated that acute normoxia causes PAEC membrane depolarization. When loaded with the nitric oxide (NO)-sensitive dye, DAF - FM, acute normoxia increased PAEC fluorescence. In PAECs derived from fetal lambs with pulmonary hypertension, an acute increase in O(2) tension had no effect on either [Ca(2+)](i) or NO production. Hypoxia increases loading of acetylcholine-sensitive calcium stores, as hypoxia potentiated the response to acetylcholine We conclude that acute normoxia increases [Ca(2+)](i) and NO production in normotensive but not hypertensive fetal PAECs via extracellular calcium entry and calcium release from calcium-sensitive intracellular stores.


Subject(s)
Calcium/metabolism , Cytosol/metabolism , Endothelial Cells/metabolism , Oxygen/metabolism , Pulmonary Artery/metabolism , Animals , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Female , Fetus/metabolism , Nitric Oxide/biosynthesis , Sheep
8.
Am J Physiol Lung Cell Mol Physiol ; 290(3): L426-33, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16467248

ABSTRACT

Pulmonary artery smooth muscle cell (PASMC) relaxation at birth results from an increase in cytosolic cGMP, cGMP-dependent and kinase-mediated activation of the Ca2+-sensitive K+ channel (KCa), and closure of voltage-operated Ca2+ channels (VOCC). How chronic intrauterine pulmonary hypertension compromises perinatal pulmonary vasodilation remains unknown. We tested the hypothesis that chronic intrauterine pulmonary hypertension selectively modifies gene expression to mitigate perinatal pulmonary vasodilation mediated by the cGMP kinase-KCa-VOCC pathway. PASMC were isolated from late-gestation fetal lambs that had undergone either ligation of the ductus arteriosus (hypertensive) or sham operation (control) at 127 days of gestation and were maintained under either hypoxic (approximately 25 Torr) or normoxic (approximately 120 Torr) conditions in primary culture. We studied mRNA levels for cGMP kinase Ialpha (PKG-1alpha), the alpha-chain of VOCC (Cav1.2), and the alpha-subunit of the KCa channel. Compared with control PASMC, hypertensive PASMC had decreased VOCC, KCa, and PKG-1alpha expression. In response to sustained normoxia, expression of VOCC and KCa channel decreased and expression of PKG-1alpha increased. In contrast, sustained normoxia had no effect on PKG-1alpha levels and an attenuated effect on VOCC and KCa channel expression in hypertensive PASMC. Protein expression of PKG-1alpha was consistent with the mRNA data. We conclude that chronic intrauterine pulmonary hypertension decreases PKG expression and mitigates the genetic effects of sustained normoxia on pulmonary vasodilation, because gene expression remains compromised even after sustained exposure to normoxia.


Subject(s)
Fetal Diseases/physiopathology , Gene Expression , Hypertension, Pulmonary/physiopathology , Muscle, Smooth, Vascular/physiology , Pulmonary Artery/physiology , Animals , Calcium/metabolism , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Calcium-Transporting ATPases , Cells, Cultured , Chronic Disease , Cyclic GMP-Dependent Protein Kinase Type I , Cyclic GMP-Dependent Protein Kinases/genetics , Cyclic GMP-Dependent Protein Kinases/metabolism , Female , Fetal Diseases/metabolism , Fetus , Hypertension, Pulmonary/metabolism , Hypoxia/metabolism , Hypoxia/physiopathology , Muscle, Smooth, Vascular/cytology , Oxygen/pharmacology , Potassium Channels/genetics , Potassium Channels/metabolism , Pregnancy , Pregnancy, Animal , Pulmonary Artery/cytology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Sheep , Vasodilation/physiology
9.
Am J Physiol Lung Cell Mol Physiol ; 290(4): L761-L768, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16284215

ABSTRACT

At birth, the lung environment changes from low to relatively high O(2) tension. Pulmonary blood flow increases and pulmonary artery pressure decreases. Recent data suggest that pulmonary vascular calcium-sensitive K(+) channel (BK(Ca)) activation mediates perinatal pulmonary vasodilation. Although BK(Ca) channel expression is developmentally regulated, the molecular mechanisms responsible for BK(Ca) expression remain unknown. We tested the hypothesis that the low-O(2) tension environment of the normal fetus modulates BK(Ca) channel expression. We analyzed BK(Ca) expression under conditions of hypoxia and normoxia both in vitro and in vivo. BK(Ca) alpha-subunit mRNA expression increased twofold in ovine pulmonary artery smooth muscle cell (PASMC) primary cultures maintained in hypoxia. In vivo, BK(Ca) expression was similarly affected by hypoxia. When adult Sprague-Dawley rats were placed in hypobaric hypoxic chambers for 3 wk, hypoxic animals showed an increase of threefold in the expression of BK(Ca) alpha- and more than twofold in the expression of BK(Ca) beta(1)-subunit mRNA. Immunochemical staining was consistent with the genetic data. To assess transcriptional activation of the beta-subunit of the BK(Ca), both BK(Ca) beta(1)- and beta(2)-subunit luciferase (K(Ca) beta:luc(+)) reporter genes were constructed. Hypoxia increased PASMC K(Ca) beta(1):luc(+) reporter expression by threefold and K(Ca) beta(2):luc(+) expression by 35%. Fetal PASMC treated with the hypoxia-inducible factor-1 mimetic deferoxamine showed a 63 and 41% increase in BK(Ca) channel alpha- and beta(1)-subunit expression, respectively. Together, these results suggest that oxygen tension modulates BK(Ca) channel subunit mRNA expression, and the regulation is, at least in part, at the transcriptional level.


Subject(s)
Hypoxia/metabolism , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Lung/physiopathology , Myocytes, Smooth Muscle/metabolism , Oxygen , Pulmonary Artery/metabolism , Animals , Calcium/metabolism , Cells, Cultured , Deferoxamine/pharmacology , Fetus/metabolism , Hypoxia/pathology , Immunohistochemistry , Intracellular Membranes/metabolism , Large-Conductance Calcium-Activated Potassium Channels/genetics , Osmolar Concentration , Partial Pressure , Peptides/pharmacology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Pulmonary Artery/pathology , Rats , Rats, Sprague-Dawley , Sheep/embryology , Transcription, Genetic
10.
Am J Physiol Lung Cell Mol Physiol ; 288(5): L917-23, 2005 May.
Article in English | MEDLINE | ID: mdl-15695541

ABSTRACT

In utero, blood shunts away from the lungs via the ductus arteriosus (DA) and the foramen ovale. After birth, the DA closes concomitant with increased oxygen tension. The present experimental series tests the hypothesis that oxygen directly increases DA smooth muscle cell (SMC) cytosolic calcium ([Ca(2+)](i)) through inactivation of a K(+) channel, membrane depolarization, and entry of extracellular calcium. To test the hypothesis, DA SMC were isolated from late-gestation fetal lambs and grown to subconfluence in primary culture in low oxygen tension (25 Torr). DA SMC were loaded with the calcium-sensitive fluorophore fura-2 under low oxygen tension conditions and studied using microfluorimetry while oxygen tension was acutely increased (120 Torr). An acute increase in oxygen tension progressively increased DA SMC [Ca(2+)](i) by 11.7 +/- 1.4% over 40 min. The effect of acute normoxia on DA SMC [Ca(2+)](i) was mimicked by pharmacological blockade of the voltage-sensitive K(+) channel. Neither removal of extracellular calcium nor voltage-operated calcium channel blockade prevented the initial increase in DA SMC [Ca(2+)](i). Manganese quenching experiments demonstrated that acute normoxia initially decreases the rate of extracellular calcium entry. Pharmacological blockade of inositol triphosphate-sensitive, but not ryanodine-sensitive, intracellular calcium stores prevented the oxygen-induced increase in [Ca(2+)](i). Endothelin increased [Ca(2+)](i) in acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca(2+)](i) via release of calcium from intracellular calcium stores, and subsequent entry of extracellular calcium, and 2) potentiates the effect of contractile agonists. Prolonged patency of the DA may result from disordered intracellular calcium homeostasis.


Subject(s)
Calcium/metabolism , Ductus Arteriosus/metabolism , Inositol Phosphates/metabolism , Muscle, Smooth/metabolism , Oxygen/metabolism , Animals , Cells, Cultured , Cytosol/metabolism , Ductus Arteriosus/cytology , Extracellular Space/metabolism , Female , Fetal Hypoxia/metabolism , Hypoxia/metabolism , Oxygen/pharmacology , Partial Pressure , Potassium Channel Blockers/pharmacology , Potassium Channels/metabolism , Pregnancy , Sheep
11.
Am J Physiol Lung Cell Mol Physiol ; 285(6): L1354-61, 2003 Dec.
Article in English | MEDLINE | ID: mdl-12882761

ABSTRACT

To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O2 sensing, fluorescence microscopy was used to study the effect of an acute increase in Po2 on the cytosolic Ca2+ concentration ([Ca2+]i) of chronically hypoxic subconfluent monolayers of PA SMC in primary culture. PA SMCs were derived from fetal lambs with PHTN due to intrauterine ligation of the ductus arteriosus. Acute normoxia decreased [Ca2+]i in control but not PHTN PA SMC. In control PA SMC, [Ca2+]i increased after Ca2+-sensitive (KCa) and voltage-sensitive (Kv) K+ channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, KCa blockade had no effect, whereas Kv blockade and diltiazem increased [Ca2+]i. Inhibition of sarcoplasmic reticulum Ca2+ ATPase activity caused a greater increase in [Ca2+]i in controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca2+]i in PHTN compared with control PA SMC. KCa channel mRNA is decreased and Kv channel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O2 sensing, alters intracellular Ca2+ homeostasis, and changes the predominant ion channel that determines basal [Ca2+]i from KCa to Kv.


Subject(s)
Fetal Diseases/physiopathology , Hypertension, Pulmonary/physiopathology , Muscle, Smooth, Vascular/physiology , Oxygen/pharmacology , Pulmonary Artery/physiology , Animals , Blood Proteins/pharmacology , Calcium/metabolism , Calcium-Transporting ATPases/antagonists & inhibitors , Cells, Cultured , Cytoplasm/metabolism , Enzyme Inhibitors/pharmacology , Female , Fetal Diseases/metabolism , Fetus , Hypertension, Pulmonary/metabolism , Hypoxia/metabolism , Hypoxia/physiopathology , Muscle, Smooth, Vascular/cytology , Peptides/pharmacology , Potassium/pharmacology , Potassium Channels/genetics , Potassium Channels/metabolism , Pregnancy , Pulmonary Artery/cytology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ryanodine/pharmacology , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Sheep , Thapsigargin/pharmacology
12.
Am J Physiol Lung Cell Mol Physiol ; 283(6): L1210-9, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12388350

ABSTRACT

K+ channels play an important role in mediating pulmonary vasodilation caused by increased oxygen tension, nitric oxide, alkalosis, and shear stress. To test the hypothesis that lung K+ channel gene expression may be altered by chronic increases in pulmonary blood flow, we measured gene and protein expression of calcium-sensitive (K Ca ) and voltage-gated (Kv2.1) K+ channels, and a pH-sensitive K+ channel (TASK), in distal lung from fetal lambs in which an aortopulmonary shunt was placed at 139 days gestation. Under baseline conditions, animals with an aortopulmonary shunt showed elevated pulmonary artery pressure and pulmonary blood flow compared with twin controls. Hypoxia caused a greater increase in pulmonary vascular tone in shunt animals compared with controls. Alkalosis caused pulmonary vasodilation in control but not shunt animals. To determine lung K+ channel mRNA levels, we performed quantitative RT-PCR. In comparison with control animals, lung K Ca channel mRNA content was increased in shunt animals, whereas TASK mRNA levels were decreased. There was no difference in Kv2.1 mRNA levels. Channel protein expression was consistent with these findings. We conclude that, in the presence of elevated pulmonary blood flow, K Ca channel expression is increased and TASK is decreased.


Subject(s)
Heart Defects, Congenital/physiopathology , Potassium Channels, Voltage-Gated , Potassium Channels/metabolism , Pulmonary Circulation/physiology , Vasomotor System/physiopathology , Alkalosis/physiopathology , Animals , Animals, Newborn/physiology , Blood Vessels/metabolism , Calcium/physiology , Delayed Rectifier Potassium Channels , Female , Fetus/physiology , Heart Defects, Congenital/embryology , Heart Defects, Congenital/metabolism , Hypertension, Pulmonary/physiopathology , Hypoxia/physiopathology , Potassium Channels/genetics , RNA, Messenger/metabolism , Shab Potassium Channels , Sheep
13.
Development ; 129(17): 4015-25, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12163405

ABSTRACT

In the early Xenopus embryo, the dorsal axis is specified by a Wnt signal transduction pathway, involving the movement of beta-catenin into dorsal cell nuclei and its functional association with the LEF-type transcription factor XTcf3. The subsequent function of XTcf3 is uncertain. Overexpression data has suggested that it can be both an activator and repressor of downstream genes. XTcf3 mRNA is synthesized during oogenesis in Xenopus and is stored in the egg. To identify its role in dorsal axis specification, we depleted this maternal store in full-grown oocytes using antisense deoxyoligonucleotides, and fertilized them. The developmental effects of XTcf3 depletion, both on morphogenesis and the expression of marker genes, show that primarily, XTcf3 is an inhibitor, preventing both dorsal and ventral cells of the late blastula from expressing dorsal genes. We also show that simple relief from the repression is not the only factor required for dorsal gene expression. To demonstrate this, we fertilized eggs that had been depleted of both XTcf3 and the maternal transcription factor VegT. Dorsal genes normally repressed by XTcf3 are not activated in these embryos. These data show that normal dorsal gene expression in the embryo requires the transcriptional activator VegT, whilst XTcf3 prevents their inappropriate expression on the ventral side of the embryo.


Subject(s)
Gene Expression Regulation, Developmental , HMGB Proteins/physiology , Organizers, Embryonic , Repressor Proteins/physiology , Transcription Factors/physiology , Xenopus Proteins , Animals , Axin Protein , Blastocyst/physiology , Body Patterning/physiology , Cytoskeletal Proteins/metabolism , Female , Goosecoid Protein , HMGB Proteins/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Nodal Signaling Ligands , Oocytes/metabolism , Proteins/genetics , Proteins/metabolism , RNA, Messenger/metabolism , Repressor Proteins/genetics , T-Box Domain Proteins/genetics , T-Box Domain Proteins/physiology , TCF Transcription Factors , Trans-Activators/metabolism , Transcription Factor 7-Like 1 Protein , Transcription Factors/genetics , Transforming Growth Factor beta/metabolism , Xenopus , beta Catenin
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