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Biomedical subjects

E Ruoslahti

Publications and source records attributed to E Ruoslahti.

At least 217 records · Page 12Linked to original sources
PubMed1983

Molecular cloning and nucleotide sequence of a cDNA clone coding for the cell attachment domain in human fibronectin.

A cDNA clone coding for the cell attachment domain in human fibronectin has been isolated using synthetic oligonucleotides. Three sets of mixed tetradecamer oligonucleotides were synthesized based on amino acid sequences in the 108-amino acid cell attachment domain (Pierschbacher, M. D., Ruoslahti, E., Sundelin, J., Lind, P., and Peterson, P. A. (1982) J. Biol. Chem. 257, 9593-9597). One of these sets was made complementary to amino acids located near the COOH terminus of the cell attachment domain and synthesized as a mixture of 24 sequences. This oligonucleotide mixture was used to prime cDNA synthesis with mRNA prepared from a human fibrosarcoma as a template. A cDNA library was constructed with the oligonucleotide-primed sequences in the vector pBR322. Colonies that hybridized with the primer were isolated from the library and further identified by hybridization with oligonucleotides deduced from an amino acid sequence located 45 amino acid residues NH2-terminal of the primer sequence. One clone which hybridized to both probes was characterized in detail. The insert was 380 base pairs long and its nucleotide sequence agreed completely with the corresponding amino acid sequence of human plasma fibronectin, showing that the sequences for this region are identical in plasma fibronectin and fibronectin from a cell line. This clone should be useful for studies on the expression of fibronectins and may also allow for the production of the biologically active cell attachment domain of fibronectin in bacteria.

Amino Acid Sequence↗
PubMed1983

Laminin from rat yolk sac tumor: isolation, partial characterization, and comparison with mouse laminin.

Laminin was isolated from a rat yolk sac tumor by salt extraction, gel filtration, and affinity chromatography on heparin-Sepharose. The purified laminin gave two polypeptide chains with approximate Mr of 200,000 and 400,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its amino acid composition and electron microscopic appearance were similar to those reported earlier for mouse laminin. Carbohydrate analysis revealed 13% carbohydrate consisting of N-acetylglucosamine, galactose, mannose, fucose, sialic acid, and small amounts of N-acetyl galactosamine. The purified rat laminin was immunologically very similar to mouse laminin as recognized by rabbit antibodies but was antigenically distinct when recognized by mouse antibodies.

Amino Acids↗
PubMed1983

Exogenous fibronectin is not required for organogenesis in vitro.

The biological effect of plasma fibronectin on the differentiation of embryonic mouse kidney and tooth was studied in organ cultures. Transferrin (50 micrograms/ml) was a strong mitogen for kidney cells, whereas the addition of soluble fibronectin (50 to 250 micrograms/ml) had no detectable effect on differentiation or proliferation. The same serum-free, transferrin-containing medium did not support tooth differentiation. However, fibronectin was not a necessary serum component because fibronectin-free serum supported tooth development. It was demonstrated with antibodies specific for human fibronectin that the exogenously added human fibronectin at 50 micrograms/ml did not become incorporated to the cultured organs. Only minimal incorporation to the kidney basement membrane area was observed when fibronectin concentration was 250 micrograms/ml. The mesenchymal stroma and the basement membranes of the kidney and tooth rudiments cultured in fibronectin-free media stained intensely with conventional fibronectin antibodies, indicating endogenous production of fibronectin. Outgrowing epithelial cells from isolated kidney tubules produced fibronectin as well as laminin. The results suggest that the fibronectin found in the stroma and basement membranes is an endogenous product of the developing tissues and that plasma fibronectin is not required for in vitro organogenesis. The results also indicate that it is difficult to study the effect of fibronectin on morphogenetic processes because it may not penetrate the organ explants in vitro.

Animals↗
PubMed1983

Immunological evidence for two distinct chondroitin sulfate proteoglycan core proteins: differential expression in cartilage matrix deficient mice.

The expression and core protein structure of two proteoglycans, the major cartilage proteoglycan isolated from a rat chondrosarcoma and a small molecular weight chondroitin sulfate proteoglycan isolated from a rat yolk sac tumor, have been compared. The cartilage proteoglycan was not detectable in the cartilage tissue of cartilage matrix deficient (cmd/cmd) neonatal mice by immunofluorescence, but the cmd cartilage did react with antibodies against the core protein of the yolk sac tumor proteoglycan. Radioimmunoassays showed that the core proteins of these proteoglycans are not cross-reactive with each other. Analysis of the core proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis after chondroitinase ABC treatment of the proteoglycan revealed a large difference in their sizes. The cartilage proteoglycan core protein had a molecular weight of about 200,000 while the yolk sac tumor proteoglycan core protein migrated with an apparent molecular weight of about 20,000. In addition, the cultured yolk sac tumor cells that make the small proteoglycan did not react with antiserum against the cartilage proteoglycan. These results indicate that the proteoglycan isolated from the yolk sac tumor is similar to the small chondroitin sulfate proteoglycan species found in cartilage and support the existence of at least two dissimilar and genetically independent chondroitin sulfate proteoglycan core proteins.

Animals↗
PubMed1983

Concurrent modulation of cell surface fibronectin and adhesion to fibronectin in hepatoma cells.

Rat hepatoma cells grown in vitro were poorly adhesive to plastic surfaces coated with fibronectin and lacked cell surface fibronectin matrix. They synthesized soluble fibronectin into the medium. The cell surface fibronectin matrix and the ability to attach to fibronectin-coated surface were restored in the 7777 cells upon passage as a tumor in rats and by coculturing these cells with normal liver-derived cells in vitro. Fibronectin matrix and the ability of cells to attach to fibronectin were thus modulated in a coordinated fashion, suggesting that the formation of a cell surface fibronectin matrix is dependent on the cell surface property that enables cells to interact with fibronectin.

Animals↗
PubMed1983

Cell adhesive, protein binding, and antigenic properties of laminin.

The cell adhesive, protein-binding and immunological properties of laminin were studied. When present on a solid-phase, laminin promoted to some degree the adhesion of various types of cells including fibroblasts, but it was less active than insolubilized fibronectin, type I collagen, or type IV collagen. When laminin was present in solution, it promoted the adhesion of cells to surfaces that would otherwise be nonadhesive. No significant binding of laminin to type IV collagen could be demonstrated by affinity assays employing radioactively labeled ligands or enzyme-labeled antibodies. Binding of laminin to anti-laminin and to polyornithine could be readily demonstrated under the same conditions. The possible autoantigenicity of laminin was studied by immunizing mice with rat and mouse laminin. Rat laminin induced antibodies that reacted strongly with rat laminin and weakly with mouse laminin in enzyme immunoassay. Mouse laminin did not induce any detectable antibodies. These results confirm earlier work on cell adhesive properties of laminin but show that laminin is different from fibronectin adhesive properties of laminin but show that laminin is different from fibronectin in that laminin promotes adhesion of cells better when presented to the cells in a soluble form. They do not support the contention that laminin would bind directly to type IV collagen or that it would be autoantigenic.

Animals↗
PubMed1983

Serum spreading factor (vitronectin) is present at the cell surface and in tissues.

Monoclonal antibodies were prepared against a cell attachment-promoting protein, serum spreading factor, which had been partially purified from human serum by chromatography on glass bead columns. The antibodies selected were those that reacted with polypeptides that had cell attachment-promoting activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunochromatography of human plasma on columns containing the monoclonal antibodies followed by affinity chromatography on heparin-Sepharose yielded material that in sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis gave polypeptides of molecular mass 65 and 75 kilodaltons. Both polypeptides bound each of three monoclonal antibodies and had cell attachment-promoting activity after transfer to nitrocellulose filters. Immunofluorescent staining of tissues with the monoclonal antibodies revealed a fibrillar pattern that was mostly associated with loose connective tissue and overlapped with fibronectin fibrils. Fetal membrane tissue, which showed strong staining with the antibodies in immunofluorescence, also gave 65- and 75-kilodalton polypeptides with cell attachment-promoting activity after chromatography on columns containing the monoclonal antibodies. One source of the tissue protein may be fibroblastic cells, because cultured human fibroblasts also stained with the monoclonal antibodies. The staining was fibrillar and appeared to be associated with the cell surface extracellular matrix. We propose the name "vitronectin" for the various forms of this protein, on the basis of its binding to glass and its adhesive properties.

Animals↗
PubMed1983

Synthetic peptide with cell attachment activity of fibronectin.

Four synthetic peptides that together constitute the cell attachment domain of fibronectin [Pierschbacher, M.D., Ruoslahti, E., Sundelin, J., Lind, P. & Peterson, P. (1982) J. Biol. Chem. 257, 9593-9597] were constructed and tested for their ability to induce cell attachment and spreading. One of these peptides, consisting of the 30 amino acid residues nearest the COOH terminus of the domain, contained all of the cell attachment activity of the whole domain. Under suitable conditions the peptide was approximately as active as intact fibronectin on a molar basis. The activity could be demonstrated by binding the peptide to polystyrene directly, or via albumin, or by coupling it to agarose beads. This synthetic peptide will be useful in the elucidation of the molecular details of the attachment of cells to fibronectin and could allow manipulation of the adhesive properties of cell culture surfaces and prosthetic materials.

Amino Acid Sequence↗
PubMed1983

Laminin promotes neuritic regeneration from cultured peripheral and central neurons.

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co-purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.

Animals↗
PubMed1983

Effect of a proteoglycan produced by rat tumor cells on their adhesion to fibronectin-collagen substrata.

In this paper, we demonstrate that a chondroitin sulfate proteoglycan purified from a rat yolk sac tumor alters the adhesion of the tumor cells to substrata containing fibronectin or type I collagen. In the presence of the proteoglycan, these substrata were much less adhesive and did not promote cell spreading. The inhibitory effect of the proteoglycan was reversible and more pronounced during the early stages of cell attachment in vitro. The effect of the proteoglycan was selective in that it depended on the ability of the adhesive substratum to bind the proteoglycan. The proteoglycan did not inhibit the attachment of the cells to type IV collagen, which bound 12 times less proteoglycan than did type I collagen. Similarly, attachment of the cells to fibronectin fragments which did not bind the proteoglycan was not affected by it. The selective effects of the proteoglycan on the adhesion of cells to extracellular matrices suggest that such proteoglycans may promote tumor invasion by reducing interaction of cells with interstitial extracellular matrices while permitting attachment to basement membranes.

Animals↗
PubMed1982

Structure of fibronectin and its fragments in electron microscopy.

Human plasma fibronectin and a series of its large proteolytic fragments were analyzed by electron microscopy using tungsten shadowing on carbon and polystyrene films. On carbon, intact fibronectin appeared as a randomly coiled strand, while on polystyrene it appeared as an elongated structure. Two fragments of fibronectin, Mr = 205000 and 190000, which lack the NH2-terminal domain of fibronectin and retain the collagen-binding, cell-attachment and heparin-binding functions, and a Mr = 170000 fragment, which retains the collagen-binding and cell-attachment functions, were seen as rods with varying degrees of nodularity while a Mr = 100000 fragment, which only binds to collagen, had two clear-cut domains. These results support the existing biochemical evidence that the segregation of the functional activities in the fibronectin molecule is based on distinct structural domains and provides evidence for the existence of an additional structural domain not revealed by biochemical and functional studies.

Chemical Phenomena↗
PubMed1982

The cell attachment domain of fibronectin. Determination of the primary structure.

The complete amino acid sequence of the cell attachment domain of human plasma fibronectin (Pierschbacher, M. D., Hayman, E. G., and Ruoslahti, E. (1981) Cell 26, 259-267) has been determined by automated sequential degradation of a peptic fragment comprising this region and of peptides derived from this fragment by digestion with thermolysin, staphylococcal V8 protease, cyanogen bromide cleavage, and partial acid hydrolysis. The fragment contains 108 residues with isoleucine and methionine as the NH2- and carboxyl-terminal amino acids, respectively. No cysteines are present. The calculated molecular weight of the cell attachment fragment, based on the amino acid sequence, is 11,482, which is in good agreement with the molecular weight estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and ultracentrifugation. There are no homologies in this fragment with other published sequences. The implications of the structure of the cell attachment fragment to the molecular mechanism of cell-fibronectin interaction are discussed.

Amino Acid Sequence↗
PubMed1982

Interactions between chondroitin sulfate proteoglycan, fibronectin, and collagen.

A proteoglycan isolated from a rat yolk sac tumor and characterized as a chondroitin sulfate proteoglycan with a smaller amount of dermatan sulfate was studied with respect to complex formation with collagen and fibronectin. The proteoglycan co-precipitated with native collagen from neutral salt solutions at 6 degrees C and 37 degrees C. Addition of fibronectin in such precipitation mixtures resulted in incorporation of fibronectin to the precipitate. Treatment of the proteoglycan with alkali to separate the glycosaminoglycan chains from the protein part and digestion of the protein part with papain greatly reduced the capacity of the proteoglycan to precipitate collagen and fibronectin. A defined extracellular matrix as represented by the complexes of collagen, proteoglycan, and fibronectin constructed here may be useful for studies on the biological effects of extracellular matrices. The multiple interactions of matrix macromolecules exemplified by these results may play a role in the formation of extracellular matrices and in the maintenance of their integrity.

Animals↗
PubMed1982

Carbohydrate structure of the concanavalin A molecular variants of alpha-fetoprotein.

The structure of the carbohydrate units of alpha-fetoprotein from fetal calf serum has been studied. Glycopeptides and oligosaccharides were prepared from alpha-fetoprotein by protease treatment and hydrazinolysis, respectively, and subjected to carbohydrate and amino acid analysis. Two N-glycosidic glycans are present in each alpha-fetoprotein molecule. These were separated into concanavalin A (ConA)-reactive and nonreactive species on ConA-Sepharose. Methylation analysis, Smith degradation, sequential exoglycosidase treatments, and sizing suggested that the major, ConA-nonreactive fraction is composed of triantennary and the minor, ConA-reactive fraction, of biantennary complex-type ConA-reactive and -nonreactive fractions of intact alpha-fetoprotein, respectively, and refractionated on Con A-Sepharose. The results indicate that 75% of alpha-fetoprotein molecules contain two triantennary complex-type glycans, 20% contain one triantennary and one biantennary glycan, and 5% contain two biantennary glycans. The last two molecular variants are bound to ConA. These results explain, at least in part, the previously found heterogeneity of alpha-fetoprotein with respect to charge and molecular size, and provide a biochemical basis for the differing reactivities toward ConA of alpha-fetoprotein from the yolk sac, fetal liver, and various tumors.

Animals↗
PubMed1982

Nonhelical, fibronectin-binding basement-membrane collagen from endodermal cell culture.

A novel method of affinity chromatography on insolubilized collagen-binding fragments of fibronectin was utilized to isolate a random-coil collagenous protein from culture media of mouse teratocarcinoma-derived endodermal cells. These cells also produced another collagenous protein, which did not bind to fibronectin but could be isolated by differential salt precipitation. The affinity-purified collagen differs from its conventionally isolated counterpart in that it is not triple-helical in structure, its polypeptides are not disulfide-crosslinked and it has affinity for fibronectin in its native state. Both collagens resemble previously characterized type IV basement-membrane collagens with respect to their amino acid composition, cyanogen bromide peptides, chain size, immunological reactivity and tissue localization. The random-coil collagen is directly active in promoting the attachment of some lines of cells, but for attachment of the endodermal cells addition of fibronectin is required. This suggests that the presence of nonhelical, fibronectin-binding collagen may have biological significance in the interaction of cells with the extracellular matrix.

Amino Acids↗