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In insects, odor cues are discriminated through a divergent family of odorant receptors (ORs). A functional OR complex consists of both a conventional odorant-binding OR and a nonconventional coreceptor (Orco) that is highly conserved across insect taxa. Recent reports have characterized insect ORs as ion channels, but the precise mechanism of signaling remains unclear. We report the identification and characterization of an Orco family agonist, VUAA1, using the Anopheles gambiae coreceptor (AgOrco) and other orthologues. These studies reveal that the Orco family can form functional ion channels in the absence of an odor-binding OR, and in addition, demonstrate a first-in-class agonist to further research in insect OR signaling. In light of the extraordinary conservation and widespread expression of the Orco family, VUAA1 represents a powerful new family of compounds that can be used to disrupt the destructive behaviors of nuisance insects, agricultural pests, and disease vectors alike.

Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a candidate gene for mediating FSHD pathophysiology, however, very little is known about the endogenous FRG1 protein. This study uses immunocytochemistry (ICC) and histology to provide insight into FRG1's role in vertebrate muscle development and address its potential involvement in FSHD pathophysiology. In cell culture, primary myoblast/myotube cultures, and mouse and human muscle sections, FRG1 showed distinct nuclear and cytoplasmic localizations and nuclear shuttling assays indicated the subcellular pools of FRG1 are linked. During myoblast differentiation, FRG1's subcellular distribution changed dramatically with FRG1 eventually associating with the matured Z-discs. This Z-disc localization was confirmed using isolated mouse myofibers and found to be maintained in adult human skeletal muscle biopsies. Thus, FRG1 is not likely involved in the initial assembly and alignment of the Z-disc but may be involved in sarcomere maintenance or signaling. Further analysis of human tissue showed FRG1 is strongly expressed in arteries, veins, and capillaries, the other prominently affected tissue in FSHD. Overall, we show that in mammalian cells, FRG1 is a dynamic nuclear and cytoplasmic protein, however in muscle, FRG1 is also a developmentally regulated sarcomeric protein suggesting FRG1 may perform a muscle-specific function. Thus, FRG1 is the only FSHD candidate protein linked to the muscle contractile machinery and may address why the musculature and vasculature are specifically susceptible in FSHD., (Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Background: Insect odorant receptors (ORs) function as odorant-gated ion channels consisting of a conventional, odorant-binding OR and the Orco coreceptor. While Orco can function as a homomeric ion channel, the role(s) of the conventional OR in heteromeric OR complexes has largely focused only on odorant recognition., Results: To investigate other roles of odorant-binding ORs, we have employed patch clamp electrophysiology to investigate the properties of the channel pore of several OR complexes formed by a range of different odorant-specific Anopheles gambiae ORs (AgOrs) each paired with AgOrco. These studies reveal significant differences in cation permeability and ruthenium red susceptibility among different AgOr complexes., Conclusions: With observable differences in channel function, the data support a model in which the odorant-binding OR also affects the channel pore. The variable effect contributed by the conventional OR on the conductive properties of odorant-gated sensory channels adds additional complexity to insect olfactory signaling, with differences in odor coding beginning with ORs on the periphery of the olfactory system.

Aedes aegypti and Anopheles gambiae are among the best-characterized mosquito species within the Culicinae and Anophelinae mosquito clades which diverged ∼150 million years ago. Despite this evolutionary distance, the olfactory systems of these mosquitoes exhibit similar morphological and physiological adaptations. Paradoxically, mosquito odorant receptors, which lie at the heart of chemosensory signal transduction pathways, belong to a large and highly divergent gene family. We have used 2 heterologous expression systems to investigate the functional characteristics of a highly conserved subset of Ors between Ae. aegypti and An. gambiae to investigate whether protein homology correlates with odorant-induced activation. We find that these receptors share similar odorant response profiles and that indole, a common and ecologically relevant olfactory cue, elicits strong responses from these homologous receptors. The identification of other highly conserved members of this Or clade from mosquito species of varying phylogenetic relatedness supports a model in which high sensitivity to indole represents an ancient ecological adaptation that has been preserved as a result of its life cycle importance. These results provide an understanding of how similarities and disparities among homologous OR proteins relate to olfactory function, which can lead to greater insights into the design of successful strategies for the control of mosquito-borne diseases.

This paper demonstrates the benefits of adopting model-based design techniques for engineering medical device software. By using a patient-controlled analgesic (PCA) infusion pump as a candidate medical device, the authors show how using models to capture design information allows for i) fast and efficient construction of executable device prototypes ii) creation of a standard, reusable baseline software architecture for a particular device family, iii) formal verification of the design against safety requirements, and iv) creation of a safety framework that reduces verification costs for future versions of the device software. 1.

Anopheles gambiae is the principal Afrotropical vector for human malaria, in which olfaction mediates a wide range of both adult and larval behaviors. Indeed, mosquitoes depend on the ability to respond to chemical cues for feeding, host preference, and mate location/selection. Building upon previous work that has characterized a large family of An. gambiae odorant receptors (AgORs), we now use behavioral analyses and gene silencing to examine directly the role of AgORs, as well as a newly identified family of candidate chemosensory genes, the An. gambiae variant ionotropic receptors (AgIRs), in the larval olfactory system. Our results validate previous studies that directly implicate specific AgORs in behavioral responses to DEET as well as other odorants and reveal the existence of at least two distinct olfactory signaling pathways that are active in An. gambiae. One system depends directly on AgORs; the other is AgOR-independent and requires the expression and activity of AgIRs. In addition to clarifying the mechanistic basis for olfaction in this system, these advances may ultimately enhance the development of vector control strategies, targeting olfactory pathways in mosquitoes to reduce the catastrophic effects of malaria and other mosquito-borne diseases., Competing Interests: The authors have declared that no competing interests exist.

In vertebrates, overexpression of facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) recapitulates the pathophysiology exhibited by FSHD patients, although the role of FRG1 in FSHD remains controversial and no precise function for FRG1 has been described in any organism. To gain insight into the function and potential role of FRG1 in FSHD, we analyzed the highly conserved Caenorhabditis elegans ortholog, frg-1. C. elegans body-wall muscles contain two distinct subcellular pools of FRG-1: nuclear FRG-1, concentrated in the nucleoli; and cytoplasmic FRG-1, associated with the Z-disk and costamere-like structures known as dense bodies. Functionally, we demonstrate that FRG-1 is an F-actin-bundling protein, consistent with its localization to dense bodies; this activity is conserved in human FRG1. This is particularly intriguing because it places FRG-1 along side the list of dense-body components whose vertebrate orthologs are involved in the myriad myopathies associated with disrupted costameres and Z-disks. Interestingly, overexpressed FRG-1 preferentially accumulates in the nucleus and, when overexpressed specifically from the frg-1 promoter, disrupts the adult ventral muscle structure and organization. Together, these data further support a role for FRG1 overexpression in FSHD pathophysiology and reveal the previously unsuspected direct involvement of FRG-1 in muscle structure and integrity.

The genetic lesion leading to facioscapulohumeral muscular dystrophy (FSHD) is a dominant deletion at the 4q35 locus. The generally accepted disease model involves an epigenetic dysregulation in the region resulting in the upregulation of one or more proximal genes whose overexpression specifically affects skeletal muscle. However, multiple FSHD candidate genes have been proposed without clear consensus. Using Xenopus laevis as a model for vertebrate development our lab has studied the effects of overexpression of the FSHD candidate gene ortholog, frg1 (FSHD region gene 1), showing that increased levels of frg1 systemically led specifically to an abnormal musculature and increased angiogenesis, the two most prominent clinical features of FSHD. Here we studied the overexpression effects of three other promising FSHD candidate genes, DUX4, DUX4c, and PITX1 using the same model system and methods for direct comparison. Expression of even very low levels of either DUX4 or pitx1 early in development led to massive cellular loss and severely abnormal development. These abnormalities were not muscle specific. In contrast, elevated levels of DUX4c resulted in no detectable adverse affects on muscle and DUX4c levels did not alter the expression of myogenic regulators. This data supports a model for DUX4 and PITX1 in FSHD only as pro-apoptotic factors if their expression in FSHD is confined to cells within the myogenic pathway; neither could account for the vascular pathology prevalent in FSHD. Taken together, increased frg1 expression alone leads to a phenotype that most closely resembles the pathophysiology observed in FSHD patients.

Background: Researchers at the Food and Drug Administration (FDA)/Center for Device and Radiological Health/Office of Science and Engineering Laboratories have been exploring the concept of model-based engineering as a means for improving the quality of medical device software. Insulin pumps were chosen as a research subject because their design provides the desired degree of research complexity and these types of devices present an ongoing regulatory challenge., Methods: Insulin pump hazards and their contributing factors are considered in the context of a highly abstract generic insulin infusion pump (GIIP) model. Hazards were identified by consulting with manufacturers, pump users, and clinicians; by reviewing national and international standards and adverse event reports collected by the FDA; and from workshops sponsored by Diabetes Technology Society. This information has been consolidated in tabular form to facilitate further community analysis and discussion., Results: A generic insulin infusion pump model architecture has been established. A fairly comprehensive hazard analysis document, corresponding to the GIIP model, is presented in this article., Conclusions: We believe that this work represents the genesis of an insulin pump safety reference standard upon which future insulin pump designs can be based to help ensure a basic level of safety. More interaction with the diabetes community is needed to assure the quality of this safety modeling process., ((c) 2010 Diabetes Technology Society.)

Diabetes Technology Society facilitated a second meeting of insulin pump experts at Mills-Peninsula Health Services, San Mateo, California on November 4, 2009, at the request of the Food and Drug Administration, Center for Devices and Radiological Health, Office of Science and Engineering Laboratories. The first such meeting was held in Bethesda, Maryland, on November 12, 2008. The group of physicians, nurses, diabetes educators, and engineers from across the United States discussed safety issues in insulin pump therapy and recommended adjustments to current insulin pump design and use to enhance overall safety. The meeting discussed safety issues in the context of pump operation; software; hardware; physical structure; electrical, biological, and chemical considerations; use; and environment from engineering, medical, nursing, and pump/user perspectives. There was consensus among meeting participants that insulin pump designs have made great progress in improving the quality of life of people with diabetes, but much more remains to be done., ((c) 2010 Diabetes Technology Society.)

Remodeling of the stromal extracellular matrix and elevated expression of specific proto-oncogenes within the adjacent epithelium represent cardinal features of breast cancer, yet how these events become integrated is not fully understood. To address this question, we focused on tenascin-C (TN-C), a stromal extracellular matrix glycoprotein whose expression increases with disease severity. Initially, nonmalignant human mammary epithelial cells (MCF-10A) were cultured within a reconstituted basement membrane (BM) where they formed three-dimensional (3-D) polarized, growth-attenuated, multicellular acini, enveloped by a continuous endogenous BM. In the presence of TN-C, however, acini failed to generate a normal BM, and net epithelial cell proliferation increased. To quantify how TN-C alters 3-D tissue architecture and function, we developed a computational image analysis algorithm, which showed that although TN-C disrupted acinar surface structure, it had no effect on their volume. Thus, TN-C promoted epithelial cell proliferation leading to luminal filling, a process that we hypothesized involved c-met, a proto-oncogene amplified in breast tumors that promotes intraluminal filling. Indeed, TN-C increased epithelial c-met expression and promoted luminal filling, whereas blockade of c-met function reversed this phenotype, resulting in normal BM deposition, proper lumen formation, and decreased cell proliferation. Collectively, these studies, combining a novel quantitative image analysis tool with 3-D organotypic cultures, demonstrate that stromal changes associated with breast cancer can control proto-oncogene function.

Paracrine signaling from lung epithelium to the surrounding mesenchyme is important for lung SMC development and function and is a contributing factor in an array of pulmonary diseases such as bronchopulmonary dysplasia, pulmonary hypertension, and asthma. Wnt7b, which is exclusively expressed in the lung epithelium, is important for lung vascular smooth muscle integrity, but the underlying mechanism by which Wnt signaling regulates lung SMC development is unclear. In this report, we have demonstrated that Wnt7b regulates a program of mesenchymal differentiation in the mouse lung that is essential for SMC development. Genetic loss-of-function studies showed that Wnt7b and beta-catenin were required for expression of Pdgfralpha and Pdgfrbeta and proliferation in pulmonary SMC precursors. In contrast, gain-of-function studies showed that activation of Wnt signaling increased the expression of both Pdgfralpha and Pdgfrbeta as well as the proliferation of SMC precursors. We further showed that the effect on Pdgfr expression was, in part, mediated by direct transcriptional regulation of the ECM protein tenascin C (Tnc), which was necessary and sufficient for Pdgfralpha/beta expression in lung explants. Moreover, this pathway was highly upregulated in a mouse model of asthma and in lung tissue from patients with pulmonary hypertension. Together, these data define a Wnt/Tnc/Pdgfr signaling axis that is critical for smooth muscle development and disease progression in the lung.

Pulmonary artery hypertension (PAH), a progressive, lethal condition that results in pathologic changes in the pulmonary arterial tree, eventually leads to right heart failure. Work identifying mutations in the Type II Bone morphogenetic protein (Bmp) receptor, BmpRII, in families with PAH has implicated Bmp-signaling in the pathogenesis of PAH. However, the effectors downstream of BmpRII in PAH remain unclear since BmpRII signals via Smad-dependent and independent mechanisms. We investigated Smad8 function, a divergent receptor regulated Smad downstream of Bmp-signaling, using gene targeting in mice. We show that Smad8 loss of function in adults resulted in characteristic changes in distal pulmonary arteries including medial thickening and smooth muscle hyperplasia that is observed in patients with PAH. Smad8 mutant pulmonary vasculature had upregulated Activin/Tgfbeta signaling and pathologic remodeling with aberrant Prx1 and Tenascin-C expression. A subset of Smad8 mutants had pulmonary adenomas uncovering a function for Smad8 in normal growth control. These findings implicate Smad8 in both pulmonary hypertension and lung tumorigenesis and support Smad8 as a candidate gene for PAH in humans.

Inflammatory processes are prominent in various types of human and experimental pulmonary hypertension (PH) and are increasingly recognized as major pathogenic components of pulmonary vascular remodeling. Macrophages, T and B lymphocytes, and dendritic cells are present in the vascular lesions of PH, whether in idiopathic pulmonary arterial hypertension (PAH) or PAH related to more classical forms of inflammatory syndromes such as connective tissue diseases, human immunodeficiency virus (HIV), or other viral etiologies. Similarly, the presence of circulating chemokines and cytokines, viral protein components (e.g., HIV-1 Nef), and increased expression of growth (such as vascular endothelial growth factor and platelet-derived growth factor) and transcriptional (e.g., nuclear factor of activated T cells or NFAT) factors in these patients are thought to contribute directly to further recruitment of inflammatory cells and proliferation of smooth muscle and endothelial cells. Other processes, such as mitochondrial and ion channel dysregulation, seem to convey a state of cellular resistance to apoptosis; this has recently emerged as a necessary event in the pathogenesis of pulmonary vascular remodeling. Thus, the recognition of complex inflammatory disturbances in the vascular remodeling process offers potential specific targets for therapy and has recently led to clinical trials investigating, for example, the use of tyrosine kinase inhibitors. This paper provides an overview of specific inflammatory pathways involving cells, chemokines and cytokines, cellular dysfunctions, growth factors, and viral proteins, highlighting their potential role in pulmonary vascular remodeling and the possibility of future targeted therapy.

The leading candidate gene responsible for facioscapulohumeral muscular dystrophy (FSHD) is FRG1 (FSHD region gene 1). However, the correlation of altered FRG1 expression levels with disease pathology has remained controversial and the precise function of FRG1 is unknown. Here, we carried out a detailed analysis of the normal expression patterns and effects of FRG1 misexpression during vertebrate embryonic development using Xenopus laevis. We show that frg1 is expressed in and essential for the development of the tadpole musculature. FRG1 morpholino injection disrupted myotome organization and led to inhibited myotome growth, while elevated FRG1 led to abnormal epaxial and hypaxial muscle formation. Thus, maintenance of normal FRG1 levels is critical for proper muscle development, supportive of FSHD disease models whereby misregulation of FRG1 plays a causal role underlying the pathology exhibited in FSHD patients. Developmental Dynamics 238:1502-1512, 2009. (c) 2008 Wiley-Liss, Inc.

The genetic lesion that is diagnostic for facioscapulohumeral muscular dystrophy (FSHD) results in an epigenetic misregulation of gene expression, which ultimately leads to the disease pathology. FRG1 (FSHD region gene 1) is a leading candidate for a gene whose misexpression might lead to FSHD. Because FSHD pathology is most prominent in the musculature, most research and therapy efforts focus on muscle cells. Previously, using Xenopus development as a model, we showed that altering frg1 expression levels systemically leads to aberrant muscle development, illustrating the potential for aberrant FRG1 levels to disrupt the musculature. However, 50-75% of FSHD patients also exhibit retinal vasculopathy and FSHD muscles have increased levels of vascular- and endothelial-related FRG1 transcripts, illustrating an underlying vascular component to the disease. To date, no FSHD candidate gene has been proposed to affect the vasculature. Here, we focus on a role for FRG1 expression in the vasculature. We found that endogenous frg1 is expressed in both the developing and adult vasculature in Xenopus. Furthermore, expression of FRG1 was found to be essential for the development of the vasculature, as a knockdown of FRG1 resulted in decreased angiogenesis and reduced expression of the angiogenic regulator DAB2. Conversely, tadpoles subjected to frg1 overexpression displayed the pro-angiogenic phenotypes of increased blood vessel branching and dilation of blood vessels, and developed edemas, suggesting that their circulation was disrupted. Thus, the systemic upregulation of the FRG1 protein shows the potential for acquiring a disrupted vascular phenotype, providing the first link between a FSHD candidate gene and the vascular component of FSHD pathology. Overall, in conjunction with our previous analysis, we show that FRG1 overexpression is capable of disrupting both the musculature and vasculature, recapitulating the two most prominent features of FSHD.

Introduction: The primary aim of this study was to evaluate the ability of radiologists to accurately estimate pneumothorax and pulmonary haemorrhage during percutaneous co-axial cutting needle CT-guided lung biopsy., Methodology: Patients undergoing cutting needle lung biopsy during the study period were identified; the path taken by the cutting needle marked on each pre-biopsy staging CT scan. Each scan was then reviewed independently by two thoracic radiologists blinded to clinical details and complications; pneumothorax and pulmonary haemorrhage risk estimated with a percentage Visual Analogue Scale., Results: In 134 patients, pneumothorax occurred in 24%. The radiologists differed in the estimation of pneumothorax risk in 55% (74 episodes). When pneumothorax risk was estimated <20% by radiologists 1 and 2, 16% and 14% of biopsies resulted in pneumothorax; where risk was estimated at 20-49%, pneumothorax incidence rose to 33% and 31%; where risk was deemed > or =50%, pneumothorax rate was 87% and 100%. Pulmonary haemorrhage occurred in 4%; estimated haemorrhage risk for biopsies complicated by haemorrhage did not differ significantly from where haemorrhage did not occur., Conclusion: Radiologists differ markedly in the estimation of pneumothorax risk for a patient undergoing co-axial lung biopsy. Identifying individual patients developing pneumothorax was only possible when risk was estimated at > or =50%. Pulmonary haemorrhage was uncommon and difficult to predict accurately.

The development of traceability methods to distinguish between farmed and wild-caught fish and seafood is becoming increasingly important. However, very little is known about how to distinguish fish originating from different farms. The present study addresses this issue by attempting to discriminate among intensively farmed freshwater Murray cod originating from different farms (indoor recirculating, outdoor floating cage, and flow through systems) in different geographical areas, using a combination of morphological, chemical, and isotopic analyses. The results show that stable isotopes are the most informative variables. In particular, delta(13)C and/or delta(15)N clearly linked fish to a specific commercial diet, while delta(18)O linked fish to a specific water source. Thus, the combination of these isotopes can distinguish among fish originating from different farms. On the contrary, fatty acid and tissue proximate compositions and morphological parameters, which are useful in distinguishing between farmed and wild fish, are less informative in discriminating among fish originating from different farms.

A burgeoning body of evidence suggests that RhoA/Rho kinase (ROCK) signalling plays an important role in the pathogenesis of various experimental models of pulmonary hypertension (PH), including chronic hypoxia-, monocrotaline-, bleomycin-, shunt- and vascular endothelial growth factor receptor inhibition plus chronic hypoxia-induced PH. ROCK has been incriminated in pathophysiologic events ranging from mediation of sustained abnormal vasoconstriction to promotion of vascular inflammation and remodelling. In addition, the 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, statins, which inhibit activation of RhoA by preventing post-translational isoprenylation of the protein and its translocation to the plasma membrane ameliorate PH in several different rat models, and may also be effective in PH patients. Also, phosphorylation of RhoA and prevention of its translocation to the plasma membrane are involved in the protective effect of the type 5-PDE inhibitor, sildenafil, against hypoxia- and bleomycin-induced PH. Collectively, these and other observations indicate that independent of the cause of PH, activation of the RhoA/ROCK pathway serves as a point of convergence of various signalling cascades in the pathogenesis of the disease. We propose that ROCK inhibitors and other drugs that inhibit this pathway might be useful in the treatment of various forms of PH.

The current goal of diabetes therapy is to reduce time-averaged mean levels of glycemia, measured as HbA1c, to prevent diabetic complications. However, HbA1c only explains <25% of the variation in risk of developing complications. Because HbA1c does not correlate with glycemic variability when adjusted for mean blood glucose, we hypothesized that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications. We show that transient hyperglycemia induces long-lasting activating epigenetic changes in the promoter of the nuclear factor kappaB (NF-kappaB) subunit p65 in aortic endothelial cells both in vitro and in nondiabetic mice, which cause increased p65 gene expression. Both the epigenetic changes and the gene expression changes persist for at least 6 d of subsequent normal glycemia, as do NF-kappaB-induced increases in monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression. Hyperglycemia-induced epigenetic changes and increased p65 expression are prevented by reducing mitochondrial superoxide production or superoxide-induced alpha-oxoaldehydes. These results highlight the dramatic and long-lasting effects that short-term hyperglycemic spikes can have on vascular cells and suggest that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications.

Age-related changes in ovarian development characteristics and plasma sex steroids in female Murray cod were examined throughout their second, third and fourth years of life to better understand the physiological and endocrine processes associated with puberty in this species in captivity. Spawning performance of 2+ and 3+ year old females was also assessed to identify ontogenetic differences in egg fertility. Puberty was acquired in 38% of 1+ year old females and 100% of age 2+ females. By age 3+, all females had developed full (adult) reproductive function. Ovarian development in pubertal fish was characterised by a rapid transition between cortical alveoli and lipid droplet oogenic phases, coinciding with significantly lower plasma 17beta-oestradiol in age 2+ females (p<0.05). Mean mature oocyte diameter (2.44 mm), post-fertilisation viability (30.80%) and hatchability (0.99%) of eggs from age 2+ females were significantly reduced relative to age 3+ adults (2.81 mm, 84.89% and 23.58%, respectively). Ovaries of pubertal Murray cod exhibited both vitellogenic and ovulatory capacities, yet functional abnormalities during secondary oocyte growth are likely to have contributed to poor egg fertility and consequently, evaluations of age-at-first maturity based on the presence of advanced ovarian stages may overestimate the reproductive potential of younger broodstock populations.

It is generally thought that mast cells influence T-cell activation nonspecifically through the release of inflammatory mediators. In this report, we provide evidence that mast cells may also affect antigen-specific T-cell responses by internalizing immunoglobulin E-bound antigens for presentation to antigen-specific T cells. Surprisingly, T-cell activation did not require that mast cells express major histocompatibility complex class II, indicating that mast cells were not involved in the direct presentation of the internalized antigens. Rather, the antigen captured by mast cells is presented by other major histocompatibility complex class II(+) antigen-presenting cells. To explore how this may occur, we investigated the fate of mast cells stimulated by antigen and found that FcepsilonRI crosslinking enhances mast cell apoptosis. Cell death by antigen-captured mast cells was required for efficient presentation because protection of mast cell death significantly decreased T-cell activation. These results suggest that mast cells may be involved in antigen presentation by acting as an antigen reservoir after antigen capture through specific immunoglobulin E molecules bound to their FcepsilonRI. This mechanism may contribute to how mast cells impact the development of T-cell responses.

The temporal dynamics of oocyte growth, plasma sex steroids and somatic energy stores were examined during a 12 month ovarian maturation cycle in captive Murray cod Maccullochella peelii peelii under simulated natural photothermal conditions. Ovarian function was found to be relatively uninhibited in captivity, with the exception that post-vitellogenic follicles failed to undergo final maturation, resulting in widespread pre-ovulatory atresia. Seasonal patterns of oocyte growth were characterised by cortical alveoli accumulation in March, deposition of lipids in April, and vitellogenesis between May and September. Two distinct batches of vitellogenic oocytes were found in Murray cod ovaries, indicating a capacity for multiple spawns. Plasma profiles of 17beta-oestradiol and testosterone were both highly variable during the maturation period suggesting that multiple roles exist for these steroids during different stages of oocyte growth. Condition factor, liver size and visceral fat stores were all found to increase prior to, or during the peak phase of vitellogenic growth. Murray cod appear to strategically utilise episodes of high feeding activity to accrue energy reserves early in the reproductive cycle prior to its deployment during periods of rapid ovarian growth.

The expanding roles of telomeres in epigenetic gene regulation, nuclear organization, and human disease have necessitated the establishment of model organisms in which to study telomere function under normal developmental conditions. We present an efficient system for generating numerous vertebrate animals containing engineered telomeres using a Xenopus laevis transgenesis technique. Our results indicate Xenopus zygotes efficiently recognize telomeric repeats at chromosome break points and form telomeric complexes thus generating a new telomere. The resulting transgenic animals progress through normal development and successfully metamorphose into froglets despite the chromosome breakage. Overall, this presents an efficient mechanism for generating engineered telomeres in a vertebrate system and provides an opportunity to investigate epigenetic aspects of telomere function during normal vertebrate development.

In amphibian oocytes, the maternal nuclear factor NF7 associates with the elongating pre-mRNAs present on the numerous lateral loops of the lampbrush chromosomes. Here, we have purified NF7 from an oocyte extract by using a combination of ion-exchange chromatography and gel filtration chromatography and demonstrated for the first time that nucleoplasmic NF7 exists primarily as free homotrimers. We confirmed the in vivo homotrimerization of NF7 by using a glutaraldehyde cross-linking assay, and we further showed that it only requires the coiled-coil domain of the NF7 tripartite motif/RBCC motif. Interestingly, we also obtained evidence that NF7 is recruited to the nucleus as a homotrimer, and expression of several mutated forms of NF7 in oocytes demonstrated that both the coiled coil and B box of NF7 are required for its chromosomal association. Together, these data strongly suggest that the interaction of NF7 with the active transcriptional units of RNA polymerase II is mediated by a trimeric B box. Finally, and in agreement with a role for NF7 in pre-mRNA maturation, we obtained evidence supporting the idea that NF7 associates with Cajal bodies.

The aim of the present investigation was to quantify the fate of C18 and long chain polyunsaturated dietary fatty acids in the freshwater fish, Murray cod, using the in vivo, whole-body fatty acid balance method. Juvenile Murray cod were fed one of five iso-nitrogenous, iso-energetic, semipurified experimental diets in which the dietary fish oil (FO) was replaced (0, 25, 50, 75, and 100%) with a blended vegetable oil (VO), specifically formulated to match the major fatty acid classes [saturated fatty acids, monounsaturated fatty acids, n-3 polyunsaturated fatty acids (PUFA), and n-6 PUFA] of cod liver oil (FO). However, the PUFA fraction of the VO was dominated by C18 fatty acids, while C20/22 fatty acids were prevalent in the FO PUFA fraction. Generally, there was a clear reflection of the dietary fatty acid composition across each of the five treatments in the carcass, fillet, and liver. Lipid metabolism was affected by the modification of the dietary lipid source. The desaturation and elongation of C18 PUFAs increased with vegetable oil substitution, supported by the occurrence of longer and higher desaturated homologous fatty acids. However, increased elongase and desaturase activity is unlikely to fulfill the gap observed in fatty acid composition resulting from decreased highly unsaturated fatty acids intake.

DNA methylation systems are well characterized in vertebrates, but methylation in Drosophila melanogaster and other invertebrates remains controversial. Using the recently sequenced honey bee genome, we present a bioinformatic, molecular, and biochemical characterization of a functional DNA methylation system in an insect. We report on catalytically active orthologs of the vertebrate DNA methyltransferases Dnmt1 and Dnmt3a and b, two isoforms that contain a methyl-DNA binding domain, genomic 5-methyl-deoxycytosine, and CpG-methylated genes. The honey bee provides an opportunity to study the roles of methylation in social contexts.

Tenascin-C (TN-C) is an extracellular matrix (ECM) protein expressed within remodeling systemic and pulmonary arteries (PAs), where it supports vascular smooth muscle cell (SMC) proliferation. Previously, we showed that A10 SMCs cultivated on native type I collagen possess a spindle-shaped morphology and do not express TN-C, whereas those on denatured collagen possess a well-defined F-actin stress fiber network, a spread morphology, and they do express TN-C. To determine whether changes in cytoskeletal architecture control TN-C, SMCs on denatured collagen were treated with cytochalasin D, which decreased SMC spreading and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling effectors required for TN-C transcription. Next, to determine whether cell shape, dictated by the F-actin cytoskeleton, regulates TN-C, different geometries of SMCs (ranging from spread to round) were engineered on denatured collagen: as SMCs progressively rounded, ERK1/2 activity and TN-C transcription declined. Because RhoA and Rho kinase (ROCK) regulate cell morphology by controlling cytoskeletal architecture, we reasoned that these factors might also regulate TN-C. Indeed, SMCs on denatured collagen possessed higher levels of RhoA activity than those on native collagen, and blocking RhoA or ROCK activities attenuated SMC spreading, ERK1/2 activity, and TN-C expression in SMCs on denatured collagen. Thus, ROCK controls the configuration of the F-actin cytoskeleton and SMC shape in a manner that is permissive for ERK1/2-dependent production of TN-C. Finally, we showed that inhibition of ROCK activity suppresses SMC TN-C expression and disease progression in hypertensive rat PAs. Thus, in addition to its role in regulating vasoconstriction, ROCK also controls matrix production.

Familial forms of human pulmonary arterial hypertension (FPAH) have been linked to mutations in bone morphogenetic protein (BMP) type II receptors (BMPR2s), yet the downstream targets of these receptors remain obscure. Here we show that pulmonary vascular lesions from patients harboring BMPR2 mutations express high levels of tenascin-C (TN-C), an extracellular matrix glycoprotein that promotes pulmonary artery (PA) smooth muscle cell (SMC) proliferation. To begin to define how TN-C is regulated, PA SMCs were cultured from normal subjects and from those with FPAH due to BMPR2 mutations. FPAH SMCs expressed higher levels of TN-C than normal SMCs. Similarly, expression of Prx1, a factor that drives TN-C transcription, was elevated in FPAH vascular lesions and SMCs derived thereof. Furthermore, Prx1 and TN-C promoter activities were significantly higher in FPAH vs. normal SMCs. To delineate how BMPR2s control TN-C, we focused on receptor (R)-Smads, downstream effectors activated by wild-type BMPR2s. Nuclear localization and phosphorylation of R-Smads was greater in normal vs. FPAH SMCs. As well, indirect blockade of R-Smad signaling with a kinase-deficient BMP receptor Ib upregulated TN-C in normal SMCs. Because ERK1/2 MAPKs inhibit the transcriptional activity of R-Smads, and because ERK1/2 promotes TN-C transcription, we determined whether ERK1/2 inhibits R-Smad signaling in FPAH SMCs and whether this activity is required for TN-C transcription. Indeed, ERK1/2 activity was greater in FPAH SMCs, and inhibition of ERK1/2 resulted in nuclear localization of R-Smads and inhibition of TN-C. These studies define a novel signaling network relevant to PAH underscored by BMPR2 mutations.

The likelihood of prevention providers and consumers adopting and implementing evidence-based HIV prevention interventions depends on the strategies employed in translating, packaging, and disseminating the findings from research to practice. Lessons from the Centers for Disease Control and Prevention's Replicating Effective Programs project have shown that to smoothly transfer HIV prevention technology from research to practice, researchers need to prepare for possible transfer during research trials. Preparation should include documenting details of the intervention beyond what is published in journals, including important details regarding what the intervention was about, how preparations for it were made, and how it was delivered. Researchers should also ensure that all relevant stakeholders are integrally involved in all aspects of the research and technology transfer process. Such collaborations encourage exchange of ideas and can make certain that interventions are designed to be relevant and acceptable to community agencies and feasible for them to implement.

The precise cellular and molecular mechanisms regulating adventitial vasa vasorum neovascularization, which occurs in the pulmonary arterial circulation in response to hypoxia, remain unknown. Here, using a technique to isolate and culture adventitial fibroblasts (AdvFBs) and vasa vasorum endothelial cells (VVECs) from the adventitia of pulmonary arteries, we report that hypoxia-activated pulmonary artery AdvFBs exhibited pro-angiogenic properties and influenced the angiogenic phenotype of VVEC, in a process of cell-cell communication involving endothelin-1 (ET-1). We demonstrated that AdvFBs, either via co-culture or conditioned media, stimulated VVEC proliferation and augmented the self-assembly and integrity of cord-like networks that formed when VVECs where cultured on Matrigel. In addition, hypoxia-activated AdvFBs produced ET-1, suggesting a paracrine role for this pro-angiogenic molecule in these processes. When co-cultured on Matrigel, AdvFBs and VVECs self-assembled into heterotypic cord-like networks, a process augmented by hypoxia but attenuated by either selective endothelin receptor antagonists or oligonucleotides targeting prepro-ET-1 mRNA. From these observations, we propose that hypoxia-activated AdvFBs exhibit pro-angiogenic properties and, as such, communicate with VVECs, in a process involving ET-1, to regulate vasa vasorum neovascularization occurring in the adventitia of pulmonary arteries in response to chronic hypoxia.

Vascular smooth muscle cells (vSMC) cultured on gels of fibrillar type I collagen or denatured collagen (gelatin) comprise a model system that has been widely used for studying the role of the extracellular matrix in vascular diseases such as hypertension, restenosis and athrosclerosis. Despite the wide use of this model system, there are several disadvantages to using collagen gels for cellular studies. These include poor optical characteristics for microscopy, difficulty in verifying that the properties of the preparations are identical from experiment to experiment, heterogeneity within the gels, and difficulty in handling the gels because they are fragile. Previously, we developed an alternative collagen matrix by forming thin films of native fibrillar collagen or denatured collagen on self-assembled monolayers of alkanethiols [Elliott, J.T., Tona, A., Woodward, J., Jones,P., Plant, A., 2003a. Thin films of collagen affect smooth muscle cell morphology. Langmuir 19, 1506-1514.]. These substrates are robust and can be characterized by surface analytical techniques that allow both verification of the reproducibility of the preparation and high-resolution analysis of collagen structure. In addition, they have excellent optical properties that allow more details of the cell-matrix interactions to be observed by microscopy. In this study, we performed a side-by-side structural and functional comparison of collagen gels with thin films of collagen. Our results indicate that vSMC on thin films of collagen are nearly identical to vSMC on thick gels as determined by morphology, proliferation rate, integrin ligation, tenascin-C expression and intracellular signaling events. These results suggest that the features of collagen gels that direct the observed vSMC responses are adequately reconstituted in the thin films of collagen. These thin films will be useful for elucidating the features of the collagen matrix that regulate vSMC response and may be applicable to high content screening.

Tenascin-C (TN-C) is a mesenchyme-derived extracellular matrix (ECM) glycoprotein required for fetal lung branching morphogenesis. Given that the low oxygen (O(2)) environment of the fetus is also essential for normal lung branching morphogenesis, we determined whether fetal O(2) tension supports this process by promoting TN-C expression. Initial studies showed that 15-day fetal rat lung explants cultured for 2 days at 3% O(2) not only branched well, but they also expressed higher levels of TN-C when compared to lungs maintained at 21% O(2), which branched poorly. Antisense oligonucleotide studies demonstrated that TN-C produced in response to 3% O(2) was essential for lung branching morphogenesis. As well, exogenous TN-C protein was shown to promote branching of lung epithelial rudiments cultured at 21% O(2). Because ECM-degrading proteinases are capable of catabolizing TN-C protein, we reasoned that 3% O(2) might promote TN-C deposition by limiting the activity of these enzymes within the fetal lung. Consistent with this idea, gelatin zymography showed that the activity of a 72-kDa gelatinase, identified as matrix metalloproteinase-2 (MMP-2), was lower at 3% O(2) vs. 21% O(2). Furthermore, pharmacologic inhibition of MMP-2 activity in fetal lung explants cultured at 21% O(2) resulted in increased TN-C deposition within the mesenchyme, as well as enhanced branching morphogenesis. Collectively, these studies indicate that fetal O(2) tension promotes TN-C-dependent lung epithelial branching morphogenesis by limiting the proteolytic turnover of this ECM component within the adjacent mesenchyme., (Copyright 2005 Wiley-Liss, Inc.)

Transcriptional repression of methylated genes can be mediated by the methyl-CpG binding protein MeCP2. Here we show that human Brahma (Brm), a catalytic component of the SWI/SNF-related chromatin-remodeling complex, associates with MeCP2 in vivo and is functionally linked with repression. We used a number of different molecular approaches and chromatin immunoprecipitation strategies to show a unique cooperation between Brm, BAF57 and MeCP2. We show that Brm and MeCP2 assembly on chromatin occurs on methylated genes in cancer and the gene FMR1 in fragile X syndrome. These experimental findings identify a new role for SWI/SNF in gene repression by MeCP2.

The Cardiff paediatric laryngoscope blade is a single blade that has been designed for use in children from birth to adolescence. This open, randomised, crossover study compared the Cardiff blade with the straight, size 1, Miller laryngoscope blade in 39 infants under 1 years of age and the curved, size 2, Macintosh blade in 39 children aged 1-16 years. The same laryngoscopic view was obtained with the Cardiff and Miller blades in 26 patients; the view was better with the Cardiff blade in seven patients and better with the Miller blade in six (median (IQR [range]) grade of laryngoscopy 1 (1-2 [1-3]) vs. 1 (1-2 [1-3]), respectively; p = 0.405). The Cardiff blade was faster at gaining a view than the Miller blade (mean (SD) time 8.5 (2.9) s vs. 10.2 (3.5) s, respectively; 95% CI for difference -2.8 to -0.4; p = 0.009). The Cardiff and Macintosh blades produced the same view in 32 patients; the view was better with the Cardiff blade in seven patients (median (IQR [range]) grade of laryngoscopy 1 (1-1 [1-3]) vs. 1 (1-2 [1-3]), respectively; p = 0.008). There was no difference in time to gain these views: mean (SD) 8.7 (3.0) s vs. 9.3 (2.7) s, respectively (95% CI for difference -1.58 to 0.40; p = 0.237). The Cardiff paediatric laryngoscope blade compares favourably with these two established laryngoscope blades in children.

DNA mutations and aberrations are a problem for all forms of life. Eukaryotes specifically have developed ways of identifying and repairing various DNA mutations in a complex and refractory chromatin environment. The chromatin structure is much more than a packaging unit for DNA; it is dynamic. Cells utilize and manipulate chromatin for gene regulation, genome organization and maintenance of genome integrity. Once a DNA aberration has occurred, the various DNA repair machineries interact with chromatin proteins, such as the histone variant H2A.X, and chromatin remodeling machines of the SWI/SNF family to gain access and repair the lesion in a timely manner. Recent studies have thus begun to address the roles of chromatin proteins in DNA repair as well as to dissect the functions of DNA repair machinery in vitro on more physiological, nucleosomal templates.

Herein, we show that the paired-related homeobox gene, Prx1, is required for lung vascularization. Initial studies revealed that Prx1 localizes to differentiating endothelial cells (ECs) within the fetal lung mesenchyme, and later within ECs forming vascular networks. To begin to determine whether Prx1 promotes EC differentiation, fetal lung mesodermal cells were transfected with full-length Prx1 cDNA, resulting in their morphological transformation to an endothelial-like phenotype. In addition, Prx1-transformed cells acquired the ability to form vascular networks on Matrigel. Thus, Prx1 might function by promoting pulmonary EC differentiation within the fetal lung mesoderm, as well as their subsequent incorporation into vascular networks. To understand how Prx1 participates in network formation, we focused on tenascin-C (TN-C), an extracellular matrix (ECM) protein induced by Prx1. Immunocytochemistry/histochemistry showed that a TN-C-rich ECM surrounds Prx1-positive pulmonary vascular networks both in vivo and in tissue culture. Furthermore, antibody-blocking studies showed that TN-C is required for Prx1-dependent vascular network formation on Matrigel. Finally, to determine whether these results were relevant in vivo, we examined newborn Prx1-wild-type (+/+) and Prx1-null (-/-) mice and showed that Prx1 is critical for expression of TN-C and lung vascularization. These studies provide a framework to understand how Prx1 controls EC differentiation and their subsequent incorporation into functional pulmonary vascular networks.

Determining how the pulmonary vascular system is formed, maintained, or disrupted during development and disease represents a major challenge in contemporary lung biology. Whereas it is appreciated that cellular proliferation, differentiation, migration, and apoptosis need to be carefully controlled in order to attain pulmonary vascular homeostasis, knowledge of the underlying cellular and molecular mechanisms involved remains surprisingly limited. Because homeobox genes represent master regulators of organogenesis and tissue patterning, it is likely that these transcription factors play a critical role in the formation of blood vessels within the lung, as well as in pathologic states in which the highly ordered structure of the pulmonary vascular tree is compromised. The aim of this review is to discuss some of the known functions of homeobox genes in the vasculature, and to extrapolate these findings to their potential roles in developing and diseased pulmonary vessels.

Platelet-derived growth factor (PDGF) inhibits expression of smooth muscle (SM) genes in vascular smooth muscle cells and blocks induction by arginine vasopressin (AVP). We have previously demonstrated that suppression of SM-alpha-actin by PDGF-BB is mediated in part through a Ras-dependent pathway. This study examined the role of phosphatidylinositol 3-kinase (PI3K)y and its downstream effector, Akt, in regulating SM gene expression. PDGF caused a rapid sustained activation of Akt, whereas AVP caused only a small transient increase. PDGF selectively caused a sustained stimulation of p85/p110 alpha PI3K. In contrast, p85/110 beta PI3K activity was not altered by either PDGF or AVP, whereas both agents caused a delayed activation of Class IB p101/110 gamma PI3K. Expression of a gain-of-function PI3K or myristoylated Akt (myr-Akt) mimicked the inhibitory effect of PDGF on SM-alpha-actin and SM22 alpha expression. Pretreatment with LY 294002 reversed the inhibitory effect of PDGF. Expression of myr-Akt selectively inhibited AVP-induced activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinases, which we have shown are critical for induction of these genes. Nuclear extracts from PDGF-stimulated or myr-Akt expressing cells showed reduced serum response factor binding to SM-specific CArG elements. This was associated with appearance of serum response factor in the cytoplasm. These data indicate that activation of p85/p110 alpha/Akt mediates suppression of SM gene expression by PDGF.

We previously demonstrated that DNA-polylactic-polyglycolic acid (PLGA)-coated stents can deliver genes to the arterial wall with reporter expression involving 1% of neointimal cells. The present study investigated a novel formulation utilizing denatured collagen in DNA-stent coatings; denatured collagen was hypothesized to enhance gene transfer due to adhesion molecule interactions and actin-related mechanisms. Arterial smooth muscle cells (SMCs) cultivated on denatured collagen had significantly greater plasmid DNA (beta-galactosidase) transfection than SMC grown on native collagen (18.3+/-1.2 vs 1.0+/-0.1%, P<0.001). The denatured-collagen effect was completely blocked with anti-alpha(v)beta(3) integrin antibody. SMCs cultivated on native collagen supplemented with tenascin-C (TN-C), a protein recognized by alpha(v)beta(3) integrins, showed a 33-fold increase in transfection compared to control (P<0.001); this effect was also blocked with anti-alpha(v)beta(3) antibody. We observed that cells grown on denatured collagen had marked F-actin-enriched stress fibers and intense perinuclear G actin, compared to those grown on native collagen, which demonstrated F-actin-enriched focal adhesions without perinuclear G-actin localization. Cytochalasin-D, an F actin depolymerizing agent, caused significantly increased SMC transfection in cells cultivated on native collagen compared to control cells (18.0+/-1.8 vs 3.02+/-0.9%, P<0.001) further supporting the view that actin-related cytoskeletal changes influence transfection. A denatured-collagen-PLGA composite vascular stent coating similarly resulted in increased plasmid DNA green fluorescent protein (GFP) expression compared to controls (P<0.001) in SMC cultures; the increased transfection was blocked by anti-alpha(v)beta(3) antibody. Pig coronary studies comparing denatured-collagen-PLGA-coated stents containing plasmid DNA (encoding GFP) to coated stents without DNA demonstrated 10.8% of neointimal cells transfected; this level of expression was almost an order of magnitude greater than previously reported with a DNA delivery stent. It is concluded that denatured collagen incorporated into plasmid DNA-stent coating formulations may increase the level of gene expression in vitro and in vivo because of integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton.

Herein, we define how MEKK1, a MAPK kinase kinase, regulates cell migration. MEKK1 is associated with actin fibers and focal adhesions, localizing MEKK1 to sites critical in the control of cell adhesion and migration. EGF-induced ERK1/2 activation and chemotaxis are inhibited in MEKK1-/- fibroblasts. MEKK1 deficiency causes loss of vinculin in focal adhesions of migrating cells, increased cell adhesion and impeded rear-end detachment. MEKK1 is required for activation of the cysteine protease calpain and cleavage of spectrin and talin, proteins linking focal adhesions to the cytoskeleton. Inhibition of ERK1/2 or calpain, but not of JNK, mimics MEKK1 deficiency. Therefore, MEKK1 regulates calpain-mediated substratum release of migrating fibroblasts.

Fibroblast migration depends, in part, on activation of FAK and cellular interactions with tenascin-C (TN-C). Consistent with the idea that FAK regulates TN-C, migration-defective FAK-null cells expressed reduced levels of TN-C. Furthermore, expression of FAK in FAK-null fibroblasts induced TN-C, whereas inhibition of FAK activity in FAK-wild-type cells had the opposite effect. Paired-related homeobox 1 (Prx1) encodes a homeobox transcription factor that induces TN-C by interacting with a binding site within the TN-C promoter, and it also promotes fibroblast migration. Therefore, we hypothesized that FAK regulates TN-C by controlling the DNA-binding activity of Prx1 and/or by inducing Prx1 expression. Prx1-homeodomain binding site complex formation observed with FAK-wild-type fibroblasts failed to occur in FAK-null fibroblasts, yet expression of Prx1 in these cells induced TN-C promoter activity. Thus, FAK is not essential for Prx1 DNA-binding activity. However, activated FAK was essential for Prx1 expression. Functionally, Prx1 expression in FAK-null fibroblasts restored their ability to migrate toward fibronectin, in a manner that depends on TN-C. These results appear to be relevant in vivo because Prx1 and TN-C expression levels were reduced in FAK-null embryos. This paper suggests a model whereby FAK induces Prx1, and subsequently the formation of a TN-C-enriched ECM that contributes to fibroblast migration.

Many nuclear hormone receptors (NHRs) actively repress the expression of their primary response genes through the recruitment of transcriptional corepressor complexes to regulated promoters. N-CoR and the highly related SMRT were originally isolated and characterized by their ability to interact exclusivelywith the unliganded forms of NHRs and confer transcriptional repression. Recently, both the N-CoR and SMRT corepressors have been found to exist in vivo in multiple, distinct macromolecular complexes. While these corepressor complexes differ in overall composition, a general theme is that they contain histone deacetylase enzymatic activity. Several of these complexes contain additional transcriptional corepressor proteins with functional ties to chromatin structure. Together, these data suggest that modulation of chromatin structure plays a central role in N-CoR mediated transcriptional repression from unliganded NHRs.

Morphogens, growth factors and extracellular matrix (ECM) components modulate early lung branching, and have been studied extensively both in vivo and in vitro. In vitro studies have been particularly useful, because tissue can be manipulated either chemically or mechanically. For the most part, such studies have been conducted at ambient oxygen tensions, despite the fact that the fetus develops in a low oxygen environment. Since oxygen tension regulates the expression of various growth factors, adhesion molecules and their receptors, we investigated whether the low oxygen environment of the fetus contributes towards lung branching morphogenesis by affecting one or more these mediators. Using an established fetal lung explant model, we demonstrated that in comparison to tissues cultured at ambient oxygen concentration (21% O2), fetal lung explants cultured at 3% O2 show increases in terminal branching and cellular proliferation, and they display appropriate proximal to distal differentiation. To investigate the factor(s) mediating the induction of lung branching morphogenesis and differentiation by fetal oxygen tension, we focused on matrix metalloproteinases (MMPs), a group of zinc-dependent enzymes that modify ECM structure and function. Our results reveal that hypoxia suppresses MMP activity, leading to the accumulation of specific ECM components, including tenascin-C (TN-C), that act to stimulate lung branching. These studies demonstrate that low oxygen in the setting of the developing lung positively regulates lung branching morphogenesis, and suggest that the pathologic responses to low oxygen in the adult lung reflect a dysregulation of this lung developmental program.

Thyroid hormone receptors (TR) act as activators of transcription in the presence of the thyroid hormone (T(3)) and as repressors in its absence. While many in vitro approaches have been used to study the molecular mechanisms of TR action, their physiological relevance has not been addressed. Here we investigate how TR regulates gene expression during vertebrate postembryonic development by using T(3)-dependent amphibian metamorphosis as a model. Earlier studies suggest that TR acts as a repressor during premetamorphosis when T(3) is absent. We hypothesize that corepressor complexes containing the nuclear receptor corepressor (N-CoR) are key factors in this TR-dependent gene repression, which is important for premetamorphic tadpole growth. To test this hypothesis, we isolated Xenopus laevis N-CoR (xN-CoR) and showed that it was present in pre- and metamorphic tadpoles. Using a chromatin immunoprecipitation assay, we demonstrated that xN-CoR was recruited to the promoters of T(3) response genes during premetamorphosis and released upon T(3) treatment, accompanied by a local increase in histone acetylation. Furthermore, overexpression of a dominant-negative N-CoR in tadpole tail muscle led to increased transcription from a T(3)-dependent promoter. Our data indicate that N-CoR is recruited by unliganded TR to repress target gene expression during premetamorphic animal growth, an important process that prepares the tadpole for metamorphosis.