Biomedical subjects

James Bristow

Publications and source records attributed to James Bristow.

12 recordsLinked to original sources

PubMed2006

Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome.

Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome-associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development.

Animals↗

PubMed2006

Application of sequence-based methods in human microbial ecology.

Ecologists studying microbial life in the environment have recognized the enormous complexity of microbial diversity for many years, and the development of a variety of culture-independent methods, many of them coupled with high-throughput DNA sequencing, has allowed this diversity to be explored in ever-greater detail. Despite the widespread application of these new techniques to the characterization of uncultivated microbes and microbial communities in the environment, their application to human health and disease has lagged. Because DNA-based techniques for defining uncultured microbes allow not only cataloging of microbial diversity but also insight into microbial functions, investigators are beginning to apply these tools to the microbial communities that abound on and within us, in what has aptly been called "the second Human Genome Project." In this review we discuss the sequence-based methods for microbial analysis that are currently available and their application to identify novel human pathogens, improve diagnosis of known infectious diseases, and advance understanding of our relationship with microbial communities that normally reside in and on the human body.

Animals↗

PubMed2005

Tenascin-X, collagen, elastin, and the Ehlers-Danlos syndrome.

Tenascin-X is an extracellular matrix protein initially identified because the gene encoding it overlaps with the human CYP21B gene. Because studies of gene and protein function of other tenascins had been poorly predictive of essential functions in vivo, we used a genetic approach that critically relied on an understanding of the genomic locus to uncover an association between inactivating tenascin-X mutations and novel recessive and dominant forms of Ehlers-Danlos syndrome (EDS). Tenascin-X provides the first example of a gene outside of the fibrillar collagens and their processing enzymes that causes EDS. Tenascin-X null mice recapitulate the skin findings of the human disease, confirming a causative role for this gene in EDS. Further evaluation of these mice showed that tenascin-X is an important regulator of collagen deposition in vivo, suggesting a novel mechanism of disease in this form of EDS. Further studies suggest that tenascin-X may do this through both direct and indirect interactions with the collagen fibril. Recent studies show that TNX effects on matrix extend beyond the collagen to the elastogenic pathway and matrix remodeling enzymes. Tenascin-X serves as a compelling example of how human "experiments of nature" can guide us to an understanding of genes whose function may not be evident from their sequence or in vitro studies of their encoded proteins.

Adult↗

PubMed2005

Gata4 expression in lateral mesoderm is downstream of BMP4 and is activated directly by Forkhead and GATA transcription factors through a distal enhancer element.

The GATA family of zinc-finger transcription factors plays key roles in the specification and differentiation of multiple cell types during development. GATA4 is an early regulator of gene expression during the development of endoderm and mesoderm, and genetic studies in mice have demonstrated that GATA4 is required for embryonic development. Despite the importance of GATA4 in tissue specification and differentiation, the mechanisms by which Gata4 expression is activated and the transcription factor pathways upstream of GATA4 remain largely undefined. To identify transcriptional regulators of Gata4 in the mouse, we screened conserved noncoding sequences from the mouse Gata4 gene for enhancer activity in transgenic embryos. Here, we define the regulation of a distal enhancer element from Gata4 that is sufficient to direct expression throughout the lateral mesoderm, beginning at 7.5 days of mouse embryonic development. The activity of this enhancer is initially broad but eventually becomes restricted to the mesenchyme surrounding the liver. We demonstrate that the function of this enhancer in transgenic embryos is dependent upon highly conserved Forkhead and GATA transcription factor binding sites, which are bound by FOXF1 and GATA4, respectively. Furthermore, the activity of the Gata4 lateral mesoderm enhancer is attenuated by the BMP antagonist Noggin, and the enhancer is not activated in Bmp4-null embryos. Thus, these studies establish that Gata4 is a direct transcriptional target of Forkhead and GATA transcription factors in the lateral mesoderm, and demonstrate that Gata4 lateral mesoderm enhancer activation requires BMP4, supporting a model in which GATA4 serves as a downstream effector of BMP signaling in the lateral mesoderm.

Animals↗

PubMed2005

Tenascin-X deficiency in autosomal recessive Ehlers-Danlos syndrome.

We present two unrelated individuals with complete deficiency of tenascin-X, resulting in an autosomal recessive form of Ehlers-Danlos syndrome (EDS). Consistent with the original description of tenascin-X deficiency, these individuals had marked skin hyperextensibility, easy bruising, and joint laxity. Unlike classical EDS they did not have atrophic scarring or poor wound healing. Significant medical problems occurring in these individuals included severe diverticular intestinal disease, mitral valve prolapse requiring valve replacement, and obstructive airway disease.

Adult↗

PubMed2004

Stem cell antigen-1 is necessary for cell-cycle withdrawal and myoblast differentiation in C2C12 cells.

Extracellular signaling pathways regulating myoblast differentiation and cell-cycle withdrawal are not completely understood. Stem cell antigen-1 (Sca-1/Ly-6A/E) is a glycosylphosphatidylinositol-anchored membrane protein known for its role in T-cell activation, and recently described as a marker for regeneration-competent myoblasts. We previously determined that expression of Sca-1/Ly-6A is transiently upregulated during myocyte cell-cycle withdrawal; however, a specific function for Sca-1 in myogenesis has not been described. Here, we show that Sca-1 expression on the surface of a subpopulation of differentiating C2C12 myoblasts is maximal at the time of cell-cycle withdrawal, and that blocking Sca-1 with monoclonal antibodies or downregulating Sca-1 expression by antisense both promotes proliferation and inhibits myotube formation. Downregulating Sca-1 expression derepresses Fyn at the time of myoblast cell-cycle withdrawal, and dominant-negative and constitutively active Fyn mutants rescue and recapitulate the Sca-1 antisense phenotype, respectively. This suggests a Fyn-mediated mechanism for Sca-1 action. Thus, we demonstrate an unprecedented role for Sca-1 in early myogenesis in C2C12 cells, and propose a novel pathway from the myoblast cell surface to intracellular signaling networks controlling proliferation versus differentiation in mammalian muscle. These findings suggest that, beyond its role as a marker for muscle progenitors, Sca-1 may be an important therapeutic target for promoting muscle regeneration.

Animals↗

PubMed2004

Formation and remodeling of the coronary vascular bed in the embryonic avian heart.

To study the formation of the coronary vessels in the developing avian heart, we stained developmentally staged quail hearts with the endothelial specific antibody QH-1. QH-1 reacted with individual cells in the proepicardial organ in Hamburger and Hamilton stage (HH) 17 embryos only after it had contacted the heart. In HH18-26 hearts, individual QH-1+ cells accumulated over the surface of the atria and ventricles. The first endothelial vessels appeared in the dorsal atrioventricular groove in HH23 hearts. CD45+ hematopoietic precursors accumulated on the heart surface, demonstrating the close temporal relationship of hematopoiesis with vasculogenesis during heart development. However, CD45 expression preceded association of these cells with the vasculature, suggesting hematopoietic commitment precedes formation of blood islands in the coronary vasculature. Endothelial tubules first appeared on the dorsal and then the ventral aspects of the heart, coalescing into large sinusoids. These sinusoids remodeled into compact muscularized vessels by HH35. Smooth muscle cell markers were first expressed at HH27 and only in association with developing vasculature. We did not observe markers of smooth muscle differentiation in the proepicardium, but it remains uncertain whether cells in the proepicardium are committed to this cell fate. Our data support a strictly vasculogenic mechanism for the formation of the coronary vessels and blood islands.

Animals↗

PubMed2003

Inhibition of alpha4-integrin stimulates epicardial-mesenchymal transformation and alters migration and cell fate of epicardially derived mesenchyme.

Epithelial-mesenchymal transformation of the embryonic epicardium produces the subepicardial mesenchyme that is essential for normal coronary vascular development. Gene targeting experiments in mice have demonstrated an essential role for alpha4-integrin in normal epicardial development, but the precise cellular consequences of alpha4-integrin loss remain uncertain. To better understand the function of alpha4-integrin in epicardial development, we constructed a replication-incompetent adenovirus (AdlacZalpha4AS) that expresses antisense chicken alpha4-integrin as the 3' untranslated region of a lacZ reporter gene. This construct effectively labeled cells while greatly reducing levels of alpha4-integrin mRNA and protein. In quail chick chimeras, transplanted epicardial cells infected with AdlacZalpha4AS adhered to the heart and were incorporated into the epicardium, but 4 days after grafting, were largely absent from the epicardial epithelium, recapitulating the defect in alpha4-null mice. This did not result from epicardial cell apoptosis or anomalous migration of epicardial cells to extracardiac sites. Rather, AdlacZalpha4AS-infected epicardial cells were particularly invasive, being three to four times more likely to migrate to the interstitium of the myocardium than AdlacZ-infected epicardial cells. Accelerated epicardial-mesenchymal transformation and migration of alpha4-negative epicardium was observed in an organ culture system that does not require prior culture of epicardial cells. Remarkably, AdlacZalpha4AS infection also prevented targeting of epicardially derived mesenchyme to the media of developing coronary vasculature in the myocardial interstitium. This study provides evidence that epicardial alpha4-integrin normally restrains epicardial-mesenchymal transformation, invasion, and migration and is essential for correct targeting of epicardially derived mesenchyme to the developing coronary vasculature.

Adenoviridae↗

PubMed2003

Genome-wide examination of myoblast cell cycle withdrawal during differentiation.

Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40% fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly-6A, whose expression specifically was up-regulated during cell cycle withdrawal coincident with early myoblast differentiation.

Animals↗

PubMed2002

Tenascin-X deficiency mimics Ehlers-Danlos syndrome in mice through alteration of collagen deposition.

Tenascin-X is a large extracellular matrix protein of unknown function. Tenascin-X deficiency in humans is associated with Ehlers-Danlos syndrome, a generalized connective tissue disorder resulting from altered metabolism of the fibrillar collagens. Because TNXB is the first Ehlers-Danlos syndrome gene that does not encode a fibrillar collagen or collagen-modifying enzyme, we suggested that tenascin-X might regulate collagen synthesis or deposition. To test this hypothesis, we inactivated Tnxb in mice. Tnxb-/- mice showed progressive skin hyperextensibility, similar to individuals with Ehlers-Danlos syndrome. Biomechanical testing confirmed increased deformability and reduced tensile strength of their skin. The skin of Tnxb-/- mice was histologically normal, but its collagen content was significantly reduced. At the ultrastructural level, collagen fibrils of Tnxb-/- mice were of normal size and shape, but the density of fibrils in their skin was reduced, commensurate with the reduction in collagen content. Studies of cultured dermal fibroblasts showed that although synthesis of collagen I by Tnxb-/- and wildtype cells was similar, Tnxb-/- fibroblasts failed to deposit collagen I into cell-associated matrix. This study confirms a causative role for TNXB in human Ehlers-Danlos syndrome and suggests that tenascin-X is an essential regulator of collagen deposition by dermal fibroblasts.

Animals↗

PubMed2002

Localization and analysis of the principal promoter for human tenascin-X.

Tenascin-X is a large extracellular matrix protein expressed in connective tissues. Mutations in TNXB are a cause of Ehlers-Danlos syndrome. Comparison of 25 kb of human and mouse DNA near the TNXB untranslated exon identified eight regions of >80% identity. Of 17 cell types and lines screened, TNXB expression was abundant only in fibroblasts and HT1080 human skin fibrosarcoma cells. Expression of TNXB promoter/reporter constructs in HT1080 cells showed that region E, near the untranslated exon, had the greatest activity, and the two regions of greatest identity, 5.0 and 3.3 kb upstream, had no activity. Mobility shift assays identified six protein-binding regions. Regions I, II, and IV bound Sp1 and Sp3, but only I and IV were functional in HT1080 cells. Regions III and V bound unknown proteins and exerted strong enhancer-like activity. Mutation of regions III and V in promoter/reporter constructs decreased TNXB transcription and identified functionally important Sp1 and Sp3 sites. These experiments provide an essential foundation for understanding the regulation of this vital protein.

Base Sequence↗

PubMed2002

Mechanisms of embryonic coronary artery development.

Coronary artery development is a complex vasculogenic process that begins shortly after heart looping. Coronary vasculogenesis is regulated by the myocardium, but is spatially and temporally dependent on the epicardium and its precursor, the proepicardial organ, for the provision of coronary vascular progenitor cells. Better understanding of the mechanisms of coronary artery development may clarify mechanisms of disease and suggest new potential therapies for disorders of the coronary vasculature.

Arteries↗