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Biomedical subjects

J R Hassell

Publications and source records attributed to J R Hassell.

At least 91 records · Page 5Linked to original sources
PubMed1986

Independence of cell shape and loss of cartilage matrix production during retinoic acid treatment of cultured chondrocytes.

Retinoic acid has been shown to cause chondrocytes in culture to flatten and to inhibit the synthesis of cartilage specific components. Since the biochemical expression of chondrocytes is considered to be dependent on cell shape, it has been proposed that retinoic acid acts on these cells primarily by causing a change in cell morphology. This hypothesis was tested by culturing chick sternal chondrocytes suspended in methyl cellulose, which prevents cell flattening. Cultures were labeled with [35S]methionine and differentiation was assessed by polyacrylamide gel electrophoresis. The results showed that retinoic acid-treated chondrocytes in suspension remained rounded but synthesized proteins characteristic of flattened or dedifferentiated chondrocytes. Chondrocytes exposed to retinoic acid in suspension became fibroblastic when placed in monolayer culture in the absence of retinoic acid. This effect was irreversible after 2 weeks of culture. These results suggest that retinoic acid has a direct molecular or biochemical effect on the chondrocyte and that the cell shape change is secondary.

Animals↗
PubMed1986

Link protein cDNA sequence reveals a tandemly repeated protein structure.

Link protein stabilizes the cartilage proteoglycan/hyaluronic acid aggregate by binding to both components. We screened a cDNA library prepared from rat chondrosarcoma mRNA in the lambda gt11 expression vector with monoclonal antibodies and polyclonal antisera to link protein. We obtained a clone for two-thirds of the link protein cDNA and identified it based on its deduced amino acid sequence. There are four RNA transcripts for link protein, ranging from 1.5 to 5.5 kilobases in size. The deduced amino acid sequence for link protein shows two domains of 100 residues each, which share 44% homology; within each domain is a 19-residue stretch 74% homologous with its counterpart. This structure indicates that link protein may have been formed by gene duplication.

Amino Acid Sequence↗
PubMed1986

Macular corneal dystrophy. Lack of keratan sulfate in serum and cornea.

An ELISA assay using a monoclonal antibody (ET-4-A-4) that recognizes a sulfated carbohydrate epitope in both keratan sulfate type I (corneal) and type II (skeletal) was employed to quantify keratan sulfate in serum and corneal tissue from patients with macular corneal dystrophy (MCD). This assay disclosed significant quantities of keratan sulfate in the serum in 45 healthy individuals (251 +/- 78 ng/ml), and in 66 patients with various corneal diseases (273 +/- 101 ng/ml). In contrast keratan sulfate was not detected (less than 2 ng/ml) in the serum of 16 patients with histopathologically confirmed MCD. Keratan sulfate was also detected in extracts of normal corneas and corneal tissue with a variety of pathologic conditions, but was virtually absent in corneal tissue from five patients with MCD. In corneas with MCD the chondroitin sulfate/keratan sulfate ratio was considerably higher than that of all normal and pathologic corneas studied. Since keratan sulfate in the serum appears to be derived predominantly from the normal turnover of cartilage these studies strongly suggest that the defect in keratan sulfate synthesis in MCD is not restricted to corneal cells and that MCD is one manifestation of a systemic disorder of keratan sulfate. The cartilage changes, however, do not have clinical significance. Moreover, since keratan sulfate can be detected in the blood of newborns it should be possible to diagnose MCD prior to corneal opacification.

Antibodies, Monoclonal↗
PubMed1985

Isolation of two forms of basement membrane proteoglycans.

Sequential extractions of the basement membrane producing Engelbreth-Holm-Swarm tumor yielded heparan sulfate proteoglycans with different size core proteins, but the same size heparan sulfate side chains. Saline, a nondenaturing solvent, extracted a small high density proteoglycan with a heterodisperse core protein of Mr = 95,000-130,000 whereas subsequent extraction with 7 M urea, a denaturing solvent, removed a large, low density proteoglycan with a Mr = 350,000-400,000 protein core. The denaturing conditions required for extraction of the large proteoglycan suggest that it interacts strongly with other basement membrane components. Antibodies to these proteoglycans cross-react with both proteoglycans, but the large proteoglycan has additional antigenic sites not present on the small proteoglycan. These proteoglycans may be derived from the same or similar gene products.

Amino Acids↗
PubMed1985

Identification of the precursor protein to basement membrane heparan sulfate proteoglycans.

The precursor protein of a basement membrane specific heparan sulfate proteoglycan has been identified as a 400,000 Mr polypeptide. Antibodies against large and small forms of this proteoglycan, isolated from a basement membrane (Engelbreth-Holm-Swarm, EHS) tumor, immunoprecipitated the same 400,000 protein from pulse-labeled EHS cells. The proteoglycan precursor protein was not recognized by antibodies against other basement membrane components or by antibodies to the cartilage proteoglycan. Furthermore, heparan sulfate proteoglycan purified from the EHS tumor blocked the immunoprecipitation of the precursor protein. Pulse-chase studies with [35S]methionine showed the precursor protein was converted to a proteoglycan. Pulse-chase studies with 35SO4 showed the large, low density proteoglycan appeared first and was degraded to a smaller, high density proteoglycan. We propose that the precursor protein is used after very little or no modification in the assembly of a large, low density heparan sulfate proteoglycan and that a portion of the population of these macromolecules are subsequently degraded to a smaller form.

Animals↗
PubMed1985

Biological activities of laminin.

Laminin is a multifunctional protein with diverse biological activities. Like fibronectin, it can influence cell adhesion, growth, morphology, differentiation, migration, and agglutination as well as the assembly of the extracellular matrix. Laminin primarily affects cells of epithelial origin, and the response varies depending on the cell. Because most differentiated cells are difficult to maintain in culture, laminin may be an important supplement in studies on cell differentiation in vitro.

Animals↗
PubMed1985

Inhibition of limb chondrogenesis by a Veratrum alkaloid: temporal specificity in vivo and in vitro.

It has been demonstrated that jervine, a steroidal alkaloid derived from plants of the genus Veratrum, exerts teratogenic effects in several animal species. Defects were restricted to structures which depend upon normal chondrogenesis for their development. Here we report studies of the temporal specificity of cellular sensitivity using limb bud mesenchyme cells obtained from Day 10 mouse embryos. These cells, when grown as high-density "spot" cultures, undergo chondrogenesis in vitro. Prior to differentiation, exposure of limb cell cultures to jervine suppressed subsequent accumulation of cartilage proteoglycans. Treatment after differentiation had no significant effect. Additionally, there was a genetic component to jervine sensitivity: C57BL/6J mice were sensitive, whereas NIH Swiss-Webster mice were insensitive. This strain-dependent difference was observed both in vivo and in vitro, supporting the validity of limb mesenchyme spot cultures as a model for jervine-induced teratogenicity. Our studies indicate that jervine acts specifically during an early phase of the differentiation of mesenchyme into cartilage. It is likely that a specific stem cell population is the target tissue of this compound.

Animals↗
PubMed1985

Heparan sulfate proteoglycans made by different basement-membrane-producing tumors have immunological and structural similarities.

Using immunological assays, we determined the relationship between the heparan sulfate proteoglycans produced by two different murine basement-membrane-producing tumors, i.e., the mouse Engelbreth-Holm-Swarm (EHS) tumor and the L2 rat yolk-sac tumor. Antibodies prepared against the heparan sulfate proteoglycans obtained from these two sources immunoprecipitated the same precursor protein with a molecular mass of 400,000 daltons from 35S-methionine pulse-labeled cells of both tumors. Immunohistochemistry showed the heparan sulfate proteoglycan to be distributed in the extracellular matrix and also in the native basement membrane of surrounding normal murine tissues. Blocking and ELISA assays demonstrated that the antibodies recognized both antigens. Using techniques involving the chemical and enzymatic degradation of 35S-sulfate-labeled glycosaminoglycans, the mouse EHS tumor cells were found to produce mainly heparan sulfate (75%) along with smaller amounts of chondroitin sulfate (19%), whereas the L2 rat yolk-sac tumor produced mainly chondroitin sulfate (76%) with smaller amounts of heparan sulfate (21%). We conclude that these two murine basement-membrane-producing tumors elaborate an immunologically and structurally similar type of high-molecular-weight heparan sulfate proteoglycan which subsequently becomes incorporated into basement-membrane-like material.

Animals↗
PubMed1984

Defective processing of keratan sulfate in macular corneal dystrophy.

Macular corneal dystrophy is a human genetic disorder characterized by corneal opacities that arise, in part, from a failure to synthesize mature keratan sulfate proteoglycans. The macromolecules in macular corneas and in keratoconus corneas, an abnormality not involving proteoglycans, were biosynthetically labeled with [3H]mannose and [14C]glucosamine in organ culture, and the keratan sulfate proteoglycans were immunoprecipitated with antibodies against the protein core of monkey keratan sulfate proteoglycan. The chondroitin sulfate proteoglycans, which did not react with the antibody, were oversulfated in corneas from patients with macular corneal dystrophy. Characterization of the immunoprecipitates showed that macular corneas did not make keratan sulfate proteoglycan but did synthesize an immunoreactive glycoprotein in nearly equal amounts as keratan sulfate proteoglycan was synthesized by the keratoconus cornea. The oligosaccharides on the immunoprecipitated macular glycoprotein appeared to be normal. However, the macromolecules contained an unsulfated glycoconjugate that was nearly as large as the normal keratan sulfate chains isolated from the keratoconus keratan sulfate-proteoglycan and contained the same relative proportions of labeled glucosamine, mannose, and fucose. This glycoconjugate was resistant to digestion with keratanase. These observations indicate that macular corneal dystrophy is caused by an error in the synthesis of keratan sulfate, possibly involving the specific sulfotransferases involved in sulfation of the lactosaminoglycan backbone of the chains.

Animals↗
PubMed1984

Retinoic acid alters the proportion of high mannose to complex type oligosaccharides on fibronectin secreted by cultured chondrocytes.

Fibronectin synthesized by retinoic acid-treated chondrocytes exhibits a higher apparent subunit molecular weight in sodium dodecyl sulfate-polyacrylamide gels than fibronectin from untreated cultures. Analyses of peptide fragments show that retinoic acid treatment alters the apparent molecular weight of the collagen-binding fragment. Approximately one-sixth of the carbohydrate moieties on the collagen-binding fragment from control chondrocytes were sensitive to endo-beta-N-acetylglucosaminidase H, whereas the collagen-binding fragment from treated chondrocytes was more resistant to endo-beta-N-acetylglucosaminidase H. These findings indicate that chondrocyte fibronectin contains oligosaccharides of the high mannose or hybrid type in contrast to fibroblast fibronectin which only contains complex type carbohydrate chains and that retinoic acid treatment results in a higher proportion of complex type carbohydrate chains on chondrocyte fibronectin.

Animals↗
PubMed1984

The regulation of basement membrane formation and cell-matrix interactions by defined supramolecular complexes.

Several constituents of basement membranes, including type IV collagen, laminin, heparan sulphate proteoglycan and nidogen, form a defined supramolecular complex that is an obligatory intermediate in the formation of this matrix. We have named this defined supramolecular complex the 'basement membrane matrisome'. Matrisome structures composed of other collagens, proteoglycans and glycoproteins may participate in the formation of other extracellular matrices. Cells show specific interactions with components of the extracellular matrix. We discuss studies that indicate that melanoma cells can express receptors for both laminin and fibronectin. However, these receptors are expressed in a reciprocal fashion, depending on the exposure of the cell to these proteins. Binding of either fibronectin or laminin to the cells elicits a distinct phenotype. This represents a mechanism in which cellular activity can be regulated by extracellular matrix factors during development and in repair.

Animals↗
PubMed1984

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells.

Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain.

Animals↗
PubMed1984

Regulation of type I collagen fibril assembly by link protein and proteoglycans.

Link protein, a glycoprotein, that is present both in cartilaginous and non-cartilaginous tissues, has previously been shown to bind to collagen and to proteoglycan. Here, we have examined the effects of link protein and proteoglycans, both alone and in combination, on the assembly of type I collagen fibrils in vitro. Link protein alone had no effect on the kinetics of fibril formation or on the size of the fibrils. Link protein, however, modulated the effects of various proteoglycans including those from bone, cartilage, cornea and sclera. Link protein had the most significant effect on fibril assembly in the presence of the low molecular weight bone proteoglycan. Although the bone proteoglycan alone had no effect on fibril formation, the fibrils were wider in the presence of link protein and proteoglycan. Cartilage proteoglycan alone increased the extent of fibril formation and the resultant fibrils were wider in diameter with a complement of incompletely assembled fibrils. In the presence of both link protein and cartilage proteoglycan, the fibrils were fully formed with the characteristic banding pattern. Further, corneal and scleral proteoglycans alone decreased the extent of fibril formation and the width of the fibrils was either unaltered or slightly decreased in the presence of the link protein. Our results indicate that both link protein and tissue-specific proteoglycans may regulate the organization of collagen fibrils in tissues.

Animals↗
PubMed1983

Interactions of basement membrane components.

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.

Animals↗
PubMed1983

Formation of a supramolecular complex is involved in the reconstitution of basement membrane components.

Basement membrane macromolecules, including type IV collagen, laminin, and heparan sulfate proteoglycan, do not aggregate when incubated alone. Rather, precipitation occurs in the presence of equimolar amounts of laminin and type IV collagen but variable amounts of heparan sulfate proteoglycan. This interaction requires native laminin and type IV collagen. Heparan sulfate proteoglycan increases the precipitation of laminin particularly in the presence of type IV collagen. Fibronectin does not cause type IV collagen to precipitate. These studies show that the components of basement membrane interact in a highly specific manner and suggest that such interactions may be involved in the deposition of basement membrane in situ.

Animals↗
PubMed1983

Proteoglycans of developing bone.

We purified and characterized the bone proteoglycans from fetal calves, growing rats, and human fetuses. The major proteoglycan is part of the mineralized tissue matrix and only 10-20% can be extracted prior to demineralization. This bone proteoglycan is a small glycoconjugate (Mr = 80,000-120,000) containing approximately 20-30% protein and either one or two chondroitin sulfate chains (Mr = 40,000) attached to a relatively monodisperse protein core (Mr = 38,000). "O"-linked and "N"-linked oligosaccharide units are also present. Antibodies directed against the protein core of calf bone proteoglycan do not cross-react with cartilage, skin, corneal, or basement membrane proteoglycans in immunoassays and have minimal cross-reactivity with scleral proteoglycans. Quantitative immunoassays and indirect immunofluorescence were used to show that the molecule is localized to forming bone trabeculae and dentin, but not to any other tissue. Osteoblasts and osteoprogenitor cells adjacent to areas undergoing rapid osteogenesis also contain this small proteoglycan. A second proteoglycan (Mr approximately equal to 1,000,000) was extracted from newly forming bone prior to demineralization. This large proteoglycan, which was isolated from the cartilage-free areas of developing intramembranous bone, has a protein core similar to that of the cartilage aggregating proteoglycan and cross-reacts with antisera raised against these cartilage proteoglycans but not with the small mineral-entrapped proteoglycan. It contains larger (Mr = 40,000) and fewer chondroitin sulfate chains than its cartilage-derived analogue, and is localized to the soft connective tissue mesenchyme lying between growing bone trabeculae. More fully formed compact bone did not contain detectable quantities of this proteoglycan.

Animals↗