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Dive into the research topics where John R. Hassell is active.

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Featured researches published by John R. Hassell.


Nature Genetics | 1999

Perlecan is essential for cartilage and cephalic development

Eri Arikawa-Hirasawa; Hiroya Takami; John R. Hassell; Yoshihiko Yamada

Perlecan, a large, multi-domain, heparan sulfate proteoglycan originally identified in basement membrane, interacts with extracellular matrix proteins, growth factors and receptors, and influences cellular signalling. Perlecan is present in a variety of basement membranes and in other extracellular matrix structures. We have disrupted the gene encoding perlecan (Hspg2) in mice. Approximately 40% of Hspg2–/– mice died at embryonic day (E) 10.5 with defective cephalic development. The remaining Hspg2–/– mice died just after birth with skeletal dysplasia characterized by micromelia with broad and bowed long bones, narrow thorax and craniofacial abnormalities. Only 6% of Hspg2 –/– mice developed both exencephaly and chondrodysplasia. Hspg2–/– cartilage showed severe disorganization of the columnar structures of chondrocytes and defective endochondral ossification. Hspg2–/– cartilage matrix contained reduced and disorganized collagen fibrils and glycosaminoglycans, suggesting that perlecan has an important role in matrix structure. In Hspg2–/– cartilage, proliferation of chondrocytes was reduced and the prehypertrophic zone was diminished. The abnormal phenotypes of the Hspg2 –/– skeleton are similar to those of thanatophoric dysplasia (TD) type I, which is caused by activating mutations in FGFR3 (refs 7, 8, 9), and to those of Fgfr3 gain-of-function mice. Our findings suggest that these molecules affect similar signalling pathways.


Experimental Eye Research | 2010

The Molecular Basis of Corneal Transparency

John R. Hassell; David E. Birk

The cornea consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells. The stroma consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25-30 nm fibrils that are regularly packed within lamellae and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sulfate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-beta would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.


Nature Genetics | 2001

Dyssegmental dysplasia, Silverman-Handmaker type, is caused by functional null mutations of the perlecan gene

Eri Arikawa-Hirasawa; William R. Wilcox; Alexander H. Le; Neil Silverman; Prasanthi Govindraj; John R. Hassell; Yoshihiko Yamada

Perlecan is a large heparan sulfate (HS) proteoglycan present in all basement membranes and in some other tissues such as cartilage, and is implicated in cell growth and differentiation. Mice lacking the perlecan gene (Hspg2) have a severe chondrodysplasia with dyssegmental ossification of the spine and show radiographic, clinical and chondro-osseous morphology similar to a lethal autosomal recessive disorder in humans termed dyssegmental dysplasia, Silverman-Handmaker type (DDSH; MIM 224410). Here we report a homozygous, 89-bp duplication in exon 34 of HSPG2 in a pair of siblings with DDSH born to consanguineous parents, and heterozygous point mutations in the 5′ donor site of intron 52 and in the middle of exon 73 in a third, unrelated patient, causing skipping of the entire exons 52 and 73 of the HSPG2 transcript, respectively. These mutations are predicted to cause a frameshift, resulting in a truncated protein core. The cartilage matrix from these patients stained poorly with antibody specific for perlecan, but there was staining of intracellular inclusion bodies. Biochemically, truncated perlecan was not secreted by the patient fibroblasts, but was degraded to smaller fragments within the cells. Thus, DDSH is caused by a functional null mutation of HSPG2. Our findings demonstrate the critical role of perlecan in cartilage development.


Archives of Biochemistry and Biophysics | 1983

Proteoglycan changes during restoration of transparency in corneal scars

John R. Hassell; Charles Cintron; Claire Kublin; David A. Newsome

Corneal scars generated in rabbits by penetrating wounds are initially opaque but become transparent within a year. Previous studies have shown that the corneal stroma consists of proteoglycans and collagen fibrils spaced at regular intervals and that the interfibrillar spaces, the presumed location of proteoglycans, are abnormally large in opaque scars. In the present study, the size and glycosaminoglycan composition of the corneal stromal proteoglycans were determined in corneal scars during the restoration of transparency. The results showed that initially opaque scars which contained the large interfibrillar spaces also contained unusually large chondroitin sulfate proteoglycans with glycosaminoglycan side chains of normal size. These opaque scars also lacked the keratan sulfate proteoglycan but did contain hyaluronic acid. In the 1-year-old scars there was a restoration of normal interfibrillar spacing, and a return to corneal stromal proteoglycans of normal size and composition. These correlations suggest that the corneal stromal proteoglycans may play a fundamental role in regulating corneal collagen fibril spacing.


Biochimica et Biophysica Acta | 1983

Interactions of basement membrane components

David T. Woodley; C. N. Rao; John R. Hassell; Lance A. Liotta; George R. Martin; Hynda K. Kleinman

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (Mr 600 000) containing the three short chains (Mr 200 000) but lacking the long chain (Mr 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.


Diabetes | 1982

Alterations in the Basement Membrane (Heparan Sulfate) Proteoglycan in Diabetic Mice

David H. Rohrbach; John R. Hassell; Hynda K. Kleinman; George R. Martin

We have grown the EHS (Engelbreth-Holm, Swarm) tumor in normal and genetically diabetic mice (db/db) and measured some components of basement membrane produced in the tumor. These studies showed similar amounts of total protein in control and diabetic tissue and similar patterns of proteins on SDS gel electrophoresis of extracts of the tissue. Laminin, a basement membrane specific glycoprotein utilized as an attachment factor by epithelial cells, was present in increased amounts in diabetic tissue. In contrast, the amount of BM-1 (heparan sulfate) proteoglycan was reduced. Less 35S-sulfate was incorporated into this proteoglycan, and the proteoglycan, but not its component glycosaminoglycans, was heterogeneous in size. The data indicate that either the synthesis of proteoglycan was decreased or its degradation was increased in diabetic tissue. Since the heparan sulfate proteoglycan serves to block the passage of anionic macromolecules through the basement membrane, decreased levels could account for the increased porosity of diabetic basement membrane. Compensatory synthesis of the basement membrane components to restore normal permeability could account for the thickened basement membranes observed in diabetes.


Developmental Biology | 1978

Inhibition of limb chondrogenesis in vitro by vitamin A: Alterations in cell surface characteristics

Cindy Arey Lewis; Robert M. Pratt; John P. Pennypacker; John R. Hassell

Abstract Mesenchyme cells derived from embryonic mouse limb buds were cultured at high cell density. During the first 24 h in culture, groups of mesenchyme cells condensed and formed cell contacts and specialized junctions. These condensations were the nodule primordia which gave rise to cartilage nodules. The cell contacts were lost as the mesenchyme cells in the primordia developed into cartilage nodules. The mature nodules contained chondrocytes isolated from one another by an extensive extracellular matrix consisting of cartilage type collagen fibrils and proteoglycan granules. The differentiation of the mesenchyme cells to chondrocytes was also characterized by the loss of a 240,000-MW cell surface glycoprotein and the appearance of an 80,000-MW surface protein. The addition of vitamin A to the medium on Day 1 inhibited chondrogenesis. The cells were closely packed together, and the limited extracellular space contained thick, banded collagen fibrils with no proteoglycan granules. The cells exhibited extensive areas of close membrane contact and specialized junctions. Vitamin A-treated cultures also retained the 240,000-MW surface glycoprotein and retarded the appearance of the 80,000-MW cell surface protein. The results of this study suggest that cell surface features normally present on mesenchyme cells are maintained and exaggerated by vitamin A.


Cell Adhesion and Communication | 1998

The Relationship between Perlecan and Dystroglycan and its Implication in the Formation of the Neuromuscular Junction

H. Benjamin Peng; A. Afshan Ali; David F. Daggett; Heikki Rauvala; John R. Hassell; Neil R. Smalheiser

Perlecan is a major heparan-sulfate proteoglycan (HSPG) within the basement membrane surrounding skeletal muscle fibers. The C-terminus of its core protein contains three globular domain modules which are also found in laminin and agrin, two proteins that bind to dystroglycan (DG, cranin) on the muscle surface with these modules. In this study, we examined whether perlecan can also bind to DG and is involved in signaling the formation of the neuromuscular junction (NMJ). By labeling cultured muscle cells with a polyclonal anti-perlecan antibody, this protein is found both within the extracellular matrix in a fibrillar network and at the cell surface in a punctate pattern. In Xenopus muscle cells, the cell-surface perlecan is precisely colocalized with DG. Both perlecan and DG are clustered at ACh receptor clusters induced by spinal neurons or by beads coated with HB-GAM, a heparin-binding growth factor. Blot overlay assays have shown that perlecan binds alpha-DG in a calcium and heparin-sensitive manner. Furthermore, perlecan is present in muscle lysate immunoprecipitated with an anti-DG antibody. Immunolabeling also showed colocalization between HB-GAM and perlecan and between HB-GAM and DG. These data suggest that perlecan is anchored to muscle surface via DG-dystrophin complex. Since DG is also a site of agrin binding, the neural agrin secreted by motoneurons during NMJ formation may compete with the pre-existing perlecan for cell surface binding. This competition may result in the presentation of perlecan-bound growth factors such as HB-GAM to effect synaptic induction.


Experimental Cell Research | 1979

The influence of an adhesive cell surface protein on chondrogenic expression in vitro.

John P. Pennypacker; John R. Hassell; Kenneth M. Yamada; Robert M. Pratt

Abstract Fibronectin is a major glycoprotein associated with fibroblasts and other cells of mesenchymal origin. However, when mesenchyme differentiates into cartilage, fibronectin is no longer synthesized. The significance of the change in fibronectin was further evaluated by culturing chondrocytes in the presence of exogenous fibronectin. Treatment with fibronectin caused the chondrocytes to assume a fibroblastic morphology and also enhanced other fibroblastic properties. These results suggest that decreased fibronectin levels may be required for chondrogenesis to occur normally.


Teratogenesis Carcinogenesis and Mutagenesis | 1982

Chondrogenesis: a model developmental system for measuring teratogenic potential of compounds.

John R. Hassell; Elizabeth A. Horigan

A simple test for determining the teratogenic potential of compounds is described using embryonic limb bud cells in culture. These mesenchyme cells multiply and differentiate into chondrocytes during a 6-day culture period. The extent of chondrogenesis is assessed by staining for the cartilage specific proteoglycan with alcian blue. The amount of stain is then measured spectrophotometrically. Compounds which interfere with growth or differentiation reduce the amount of proteoglycan and as a consequence, reduce alcian blue staining. Compounds can be added directly to the media or be activated using several different metabolizing systems. The dose of a compound needed to reduce alcian blue staining by 50% is designated the teratogenic potential (TP50) of that compound. TP50s of proven teratogens compare favorably with in vivo teratogenic doses of the teratogens.

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Bradley Kane

University of South Florida

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David A. Newsome

Louisiana State University

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Hynda K. Kleinman

National Institutes of Health

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George R. Martin

National Institutes of Health

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Y Yamada

National Institutes of Health

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David E. Birk

University of South Florida

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Elizabeth A. Horigan

National Institutes of Health

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Kurt Musselmann

University of South Florida

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Shukti Chakravarti

Johns Hopkins University School of Medicine

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