IntroductionHyaluronan, or hyaluronic acid (HA) is a linear, highmolecular-weight (mega-Dalton) polymer comprised of repeating disaccharide units of (β1→3)D-glucuronate-(β1→4)N-acetyl-D-glucosamine. HA is synthesized by integral plasma membrane glycosyltransferases and is exported directly into the extracellular space (1, 2). Although HA is chemically homogeneous, there are three distinct mammalian HA synthases (designated Has1, Has2, and Has3), encoded by related but nonlinked genes (3-9). Each synthase has distinct catalytic properties, and the distribution and abundance of each varies during development of the mouse (6, 10). These observations suggest that the different Has enzymes play distinct roles.HA binds salt and water, expanding the extracellular space (11)(12)(13)(14). HA is especially prominent at sites where cell migration occurs, such as pathways of neural crest cell migration and in the developing cardiovascular system. In vivo, HA interacts with other extracellular matrix molecules, typically via an HA-binding domain called the link module (15). These interactions create a supramolecular architecture of the extracellular matrix, i.e., the composite matrix network of HA, link protein, and aggrecan that plays a critical role in load-bearing articular cartilage (16)(17)(18).In addition to its important physical properties, the overexpression of Has genes results in increased anchorage-independent growth and metastasis of transformed cells (19,20), suggesting a link between HA and transformation. HA is also implicated in receptor-mediated cell adhesion and intracellular signaling (21,22). Taken together, such observations suggest that HA plays a vital role in diverse cellular events, including cell migration, tissue remodeling, and metastasis. However, the near-ubiquitous distribution of HA in vivo, the biological activity of HA fragments released by degradative enzymes (23), and the inability to inhibit HA synthesis in vivo have hindered definitive analysis of the physiological roles of HA. Accordingly, we used a genetic approach to investigate the roles of HA in vivo and to identify the HA synthase that is critical during embryogenesis.Expression of Has2 appeared to correlate with expansion of cardiac cushion tissue and subsequent transformation of endocardial cells into mesenchyme. The tar- We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2 -/-embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2 -/-embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2 -/-explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, ...
Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K m values for the two substrates UDPGlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 ؋ 10 5 to 1 ؋ 10 6 Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 ؋ 10 5 to ϳ2 ؋ 10 6 Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 ؋ 10 5 to ϳ2 ؋ 10 6 Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 ؋ 10 6 Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions. Hyaluronan (HA)1 is a major component of most extracellular matrices, particularly in tissues with rapid cell proliferation and cell migration (1). The interaction of HA with various HA-binding proteins and cell-surface receptors plays important roles in regulating fundamental cell behaviors such as cell adhesion, migration, and differentiation (2, 3). Thus, HA has been greatly implicated in morphogenesis, regeneration, wound healing, tumor invasion, and cancer metastasis (4 -6). In addition, HA is an important structural molecule required for the maintenance of various aspects of tissue architecture and function. The physical and biological properties of HA appear to be affected by many factors including HA concentration and chain length. Indeed, high molecular weight HA at high concentrations suppresses endothelial cell growth, whereas low molecular weight HA stimulated cell growth leading to induction of angiogenesis (7). In addition, viscosity of the HA gel and the ability to hydrate large amounts of water were shown to be dependent on the molecular size of the HA chain.HA is a high molecular weight linear polymer composed of GlcUA -1,3-GlcNAc -1,4 disaccharide units and is synthesized by HA synthase at the inner face of the plasma membrane (8). Although a great deal of effort has been made to elucidate the mechanism of HA biosynthesis in mammalian cells, it has remained unclear due to difficulty in biochemical isolation of the active enzyme (9 -11). Recently, three distinct yet highly conserved genes encoding mammali...
We present a method for solving the following problem: Given a set of data points scattered in three dimensions and an initial triangular mesh M0, produce a mesh M, of the same topological type as M0, that fits the data well and has a small number of vertices. Our approach is to minimize an energy function that explicitly models the competing desires of conciseness of representation and fidelity to the data. We show that mesh optimization can be effectively used in at least two applications: surface reconstruction from unorganized points, and mesh simplification (the reduction of the number of vertices in an initially dense mesh of triangles).
Idiopathic pulmonary fibrosis is an inexorably fatal disorder characterized by connective tissue deposition within the terminal air spaces resulting in loss of lung function and eventual respiratory failure. Previously, we demonstrated that foci of activated fibroblasts expressing high levels of fibronectin, procollagen, and smooth muscle actin and thus resembling those found in healing wounds are responsible for the connective tissue deposition and scarring in idiopathic pulmonary fibrosis. Using in situ hybridization and immunohistochemistry, we now demonstrate the presence of transforming growth factor beta 1 (TGF-beta 1), a potent profibrotic cytokine, in the foci containing these activated fibroblasts. These results suggest that matrix-associated TGF-beta 1 may serve as a stimulus for the persistent expression of connective tissue genes. One potential source of the TGF-beta 1 is the alveolar macrophage, and we demonstrate the expression of abundant TGF-beta 1 mRNA in alveolar macrophages in lung tissue from patients with idiopathic pulmonary fibrosis.
have identified a new fibronectin receptor that is identical to the integrin receptor a4B1. mAbs P3E3, P4C2, and P4G9 recognized epitopes on the tx4 subunit and completely inhibited the adhesion of peripheral blood and cultured T lymphocytes to a 38-kD tryptic fragment of plasma fibronectin containing the carboxy-terminal Heparin II domain and part of the type III connecting segment (IIICS). The ligand in IrlCS for ot4B1 was the CS-1 region previously defined as an adhesion site for melanoma cells. The functionally defined mAbs to or4 partially inhibited T lymphocyte adhesion to intact plasma fibronectin and had no effect on their attachment to an 80-kD tryptic fragment containing the RGD (arg-glyasp) adhesion sequence, mAbs (P1D6 and P1F8) to the previously described fibronectin receptor, ot5B1, completely inhibited T lymphocyte adhesion to the 80-kD fragment but had no effect on their attachment to the 38-kD fragment or to CS-1. Both ot4/~l and ot5/31 localized to focal adhesions when fibroblasts that express these receptors were grown on fibronectin-coated surfaces. These findings demonstrated a specific interaction of both receptors with fibronectin at focal contacts.In conclusion, these findings show clearly that cultured T lymphocytes use two independent receptors during attachment to fibronectin and that (a) ~x5~l is the receptor for the RGD containing cell adhesion domain, and (b) ot4~l is the receptor for a carboxyterminal cell adhesion region containing the Heparin II and IIICS domains. Furthermore, these data also show that T lymphocytes express a clear preference for a region of molecular heterogeneity in IIICS (CS-1) generated by alternative splicing of fibronectin pre-mRNA and that o~4~1 is the receptor for this adhesion site.
We present a general method for automatic reconstruction of accurate, concise, piecewise smooth surface models from scattered range data. The method can be used in a variety of applications such as reverse engineering -the automatic generation of CAD models from physical objects. Novel aspects of the method are its ability to model surfaces of arbitrary topological type and to recover sharp features such as creases and corners. The method has proven to be effective, as demonstrated by a number of examples using both simulated and real data.A key ingredient in the method, and a principal contribution of this paper, is the introduction of a new class of piecewise smooth surface representations based on subdivision. These surfaces have a number of properties that make them ideal for use in surface reconstruction: they are simple to implement, they can model sharp features concisely, and they can be fit to scattered range data using an unconstrained optimization procedure.
Abstract-Recent evidence has identified the peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1␣) as a regulator of cardiac energy metabolism and mitochondrial biogenesis. We describe the development of a transgenic system that permits inducible, cardiac-specific overexpression of PGC-1␣. Expression of the PGC-1␣ transgene in this system (tet-on PGC-1␣) is cardiac-specific in the presence of doxycycline (dox) and is not leaky in the absence of dox.Overexpression of PGC-1␣ in tet-on PGC-1␣ mice during the neonatal stages leads to a dramatic increase in cardiac mitochondrial number and size coincident with upregulation of gene markers associated with mitochondrial biogenesis. In contrast, overexpression of PGC-1␣ in the hearts of adult mice leads to a modest increase in mitochondrial number, derangements of mitochondrial ultrastructure, and development of cardiomyopathy. The cardiomyopathy in adult tet-on PGC-1␣ mice is characterized by an increase in ventricular mass and chamber dilatation. Surprisingly, removal of dox and cessation of PGC-1␣ overexpression in adult mice results in complete reversal of cardiac dysfunction within 4 weeks. These results indicate that PGC-1␣ drives mitochondrial biogenesis in a developmental stage-dependent manner permissive during the neonatal period. This unique murine model should prove useful for the study of the molecular regulatory programs governing mitochondrial biogenesis and characterization of the relationship between mitochondrial dysfunction and cardiomyopathy and as a general model of inducible, reversible cardiomyopathy. Key Words: mitochondria Ⅲ metabolism Ⅲ transgenic mice Ⅲ cardiomyopathy Ⅲ transcription factors T he heart has an extraordinarily high capacity for mitochondrial ATP production to meet the rigorous and dynamic energy demands of the postnatal environment. The importance of the mitochondrion for cardiac function is underscored by the development of cardiomyopathy in inherited and acquired forms of mitochondrial dysfunction in humans. [1][2][3] Because mitochondria contain proteins encoded by both nuclear and mitochondrial genes, mitochondrial biogenesis requires the coordinate regulation of these 2 genomes. The transcriptional regulatory network controlling the expression of nuclear and mitochondrial genes includes nuclear respiratory factor (NRF)-1 and NRF-2 and mitochondrial transcription factor A (mtTFA). The regulatory pathways upstream of these factors are a focus of intense investigation. 4 -7 Recently, the peroxisome proliferator-activated receptor (PPAR) ␥ coactivator-1␣ (PGC-1␣) has been identified as an inducible upstream regulator of mitochondrial number and function. 8 -10 PGC-1␣ is a transcriptional coactivator that lacks DNA-binding activity but interacts with and coactivates numerous transcription factors, including nuclear receptors such as PPAR␥ and PPAR␣, estrogen receptor ␣, thyroid hormone, retinoid receptors, and hepatocyte nuclear factor-4␣. 8,11-16 PGC-1␣ also coactivates nonnuclear receptor transcription factors, ...
Fibronectin (Fn) matrices are vital to vertebrate development and wound healing and modulate tumorigenesis. We used a recombinant Fn-binding integrin alpha IIb beta3, to define rules for integrin-initiated Fn matrix formation. We report the following. First, multiple Fn-binding integrins can support matrix assembly; their activation state controls fibrillogenesis. Second, Fn binding to cells expressing an activated integrin is necessary but not sufficient for matrix assembly. Additional "postoccupancy" events involving the integrin beta, but not the alpha subunit, cytoplasmic domain are needed. Third, these postoccupancy events require an intact actin cytoskeleton. We propose a model for integrin involvement in Fn fibrillogenesis that reconciles previous paradoxes and suggests novel approaches to the therapeutic control of Fn matrix assembly.
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