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Biomedical subjects

E Ruoslahti

Publications and source records attributed to E Ruoslahti.

At least 235 records · Page 13Linked to original sources
PubMed1982

Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells.

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.

Animals↗
PubMed1982

Cell attachment on replicas of SDS polyacrylamide gels reveals two adhesive plasma proteins.

A novel procedure that detects adhesive proteins in complex mixtures was used to characterize such proteins in plasma. The proteins are separated by SDS PAGE and transferred to nitrocellulose filters. Cells incubated on these filters attach to those proteins that have adhesive properties. When applied to human plasma proteins this procedure reveals, in addition to fibronectin, a cell-attachment protein with a polypeptide molecular weight of 70,000. Using a monoclonal antibody that inhibits attachment of cells to fibronectin, we show that this polypeptide is not a fragment of fibronectin and we present evidence that it is a component of the serum spreading factor. Therefore, as defined by our assay, this protein and fibronectin are the major attachment proteins for fibroblastic cells in plasma or serum.

Animals↗
PubMed1982

Molecular and biological interactions of fibronectin.

Fibronectin is a high molecular weight glycoprotein present at cell surfaces and in various body fluids. It is involved in cellular adhesion. The frequent absence of fibronectin from the surface of malignant cells may contribute to the invasive growth of tumors. To develop a better understanding of the functions of fibronectin, we have studied its structure by making use of proteolytic fragmentation and monoclonal antibodies. Fragments of plasma fibronectin which retain one or several of the affinities of intact fibronectin have been purified using affinity chromatography on collagen and heparin, and on monoclonal antibodies. These studies reveal a structure in which the different functions are located in separate protease-resistant molecular domains. The NH2-terminal domain contains the binding sites for fibrin, actin, and Staphylococci; this is followed by a collagen-binding domain, the cell attachment domain, and at the COOH-terminal end, the heparin-binding domain. These binding sites allow fibronectin to participate in multiple interactions with collagens and proteoglycans. The insoluble complexes formed by these 3 components that can be generated in vitro may represent a model for the basic structure of extracellular matrices. The role of fibronectin in such a matrix may be to contribute to the stability of the complex and to provide adhesion sites for cells. Disturbances in the interactions involving fibronectin could be potentially important to understanding diseases of connective tissues and in malignancy.

Animals↗
PubMed1982

Unfolded transferrin polypeptide chain is immunologically crossreactive with similar derivatives of serum albumin and alpha-fetoprotein.

Radioimmunoassays utilizing reduced and carboxymethylated (RC) proteins as antigens reveal a cross-reactivity between alpha-fetoprotein (AFP) and albumin. Similar assays were used to study the relationships of AFP and albumin to other serum proteins. Of the several serum proteins tested, transferrin showed the most similarity with AFP and albumin. There was no cross-reactivity of the native proteins, but antisera prepared against RC-albumin and RC-AFP bound 125I-labeled RC-transferrin at high titers, and antiserum to RC-transferrin bound labeled RC-AFP but not RC-albumin. Inhibition assays utilizing binding of 125I-RC-AFP or 125I-RC-transferrin to anti-RC-albumin showed that the RC derivatives of AFP, albumin, and transferrin were equally efficient inhibitors, whereas other serum proteins inhibited much less. The serum vitamin D carrier protein (Gc protein) showed intermediate reactivity. The reactivities of the antisera to RC-albumins with RC-transferrin and RC-Gc protein were corroborated by immunostaining of proteins separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. These antisera stained the bands formed by RC derivatives of albumin, AFP, transferrin, and Gc protein, but not other proteins tested. AFP and albumin are known to have amino acid sequence homology. Our results suggest that transferrin and possibly also Gc protein may be structurally related to AFP and albumin.

Amino Acid Sequence↗
PubMed1982

Expression of fibronectin and laminin in the rat liver after partial hepatectomy, during carcinogenesis, and in transplantable hepatocellular carcinomas.

The distribution in the F344 rat liver of two extracellular matrix and basement membrane components, fibronectin and laminin, was studied by immunofluorescence. Fibronectin was found diffusely in normal liver lining the sinusoids and in connective tissue surrounding blood vessels and bile ducts; laminin was present predominantly in the basement membranes of blood vessels and bile ducts and was only inconsistently seen lining the sinusoids. After partial hepatectomy (PH), there was a transient decrease of fibronectin in the central and midzone sinusoidal hepatic areas. This decrease was most marked on day 3 after the PH. Carcinogens caused marked changes in the distribution of fibronectin. Large extracellular deposits of fibronectin were seen in areas of oval cell proliferation in livers of rats treated with N-2-fluorenylacetamide (2-FAA) while being fed a choline-deficient diet. In contrast, the nodules that developed in these livers were almost completely devoid of fibronectin staining. Neoplastic nodules produced in rats by cyclic feedings of 2-FAA r by injections of diethylnitrosamine also contained little or no fibronectin. Laminin staining did not change markedly during these treatments, but increased staining was seen associated with the newly formed ductlike structures and oval cells in liver of rats treated with carcinogens. Transplantable hepatomas varied in their fibronectin staining from fibronectin-negative hepatomas to ones with fibronectin staining within or around every tumor cell. Laminin was only found around the vascular structures within the tumors. The presence or absence of fibronectin in hepatomas did not show an obvious correlation to growth rate or metastatic potential of the tumors studied.

2-Acetylaminofluorene↗
PubMed1982

Characterization and use of an allotype-specific monoclonal antibody to placental alkaline phosphatase in the study of cancer-related phosphatase polymorphism.

The hybridoma technique was used to produce an allotype-specific monoclonal antibody (F11) that reacts with the products of the S, I, and D alleles of PLAP but not of the F allele. Serum and ascites samples from patients with different cancers containing high levels of PLAP were tested for reactivity with F11. These tumor-derived PLAPs were of the Nagao type as shown by their sensitivity to inhibition by L-leucine. This type of inhibition is exhibited also by the rare D allelic variant of PLAP but not by the common forms. Thus, it has been proposed that the Nagao enzyme represents reexpression of the D allele of PLAP. F11 reactive and nonreactive samples as well as samples with intermediate reactivity were found among the cancer sera and ascites. Our results show tha tumor-derived Nagao enzyme does not represent the reexpression of the D allele but instead, in spite of its distinct inhibition pattern, expresses the same genetic polymorphism that is found in the placenta.

Alkaline Phosphatase↗
PubMed1982

Placental alkaline phosphatase as a tumor marker for seminoma.

A sensitive and specific enzyme-linked immunoabsorbent assay was used in a retrospective study of serum levels of placental alkaline phosphatase (PLAP) in testicular cancer. Sixteen of 28 men with active seminoma had elevated PLAP levels, and 71% had elevated levels of either PLAP, human chorionic gonadotropin, or both. Only four of 22 men with active nonseminomatous cancer had elevated PLAP levels, and the levels were normal in all control patients, including 33 men apparently cured of testicular cancer. In six of ten serial studies, PLAP levels provided information not otherwise available that would have been useful clinically, and the levels never were elevated inappropriately. Our data suggest that PLAP is a clinically useful serum tumor marker for seminoma.

Alkaline Phosphatase↗
PubMed1981

Isolation of a chondroitin sulfate proteoglycan from a rat yolk sac tumor and immunochemical demonstration of its cell surface localization.

A proteoglycan was isolated from ascites fluid produced by a rat yolk sac tumor. The glycosaminoglycan chains of the proteoglycan are all sensitive to digestion with chondroitinase ABC and about 90% are sensitive to chondroitinase AC. The proteoglycan contains 5% protein. Amino acid analysis revealed a high content of serine and glycine which together constitute 37% of the amino acids. Glutamic acid (glutamine) and aspartic acid (asparagine) are also abundant. Galactosamine accounts for 97% of the hexosamine and the remainder is glucosamine. These characteristics indicate that the glycosaminoglycan side chains of this proteoglycan are predominantly chondroitin sulfate with a smaller amount of dermatan sulfate. Antibodies to the proteoglycan were prepared by immunization of a rabbit with purified alkali-treated proteoglycan. Affinity-purified antibodies from the antiserum immunoprecipitated (35S)sulfate-labeled radioactivity from culture media of the yolk sac tumor cells known to contain chondroitin sulfate proteoglycan. This binding was inhibited by the intact purified proteoglycan but not by proteoglycan treated with papain, suggesting dependence of the reactivity of the antibodies on integrity of the protein part of the proteoglycan. Immunofluorescence of the cultured yolk sac tumor cells revealed localization of immune reactive proteoglycans at the cell surface.

Amino Acids↗
PubMed1981

Alignment of biologically active domains in the fibronectin molecule.

Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.

Amino Acid Sequence↗
PubMed1981

Fibronectin from human germ-cell tumors resembles amniotic fluid fibronectin.

Three human germ-cell tumors established as xenografts in immunosuppressed mice were shown to produce fibronectin. Immunoperoxidase tissue staining with a specific antiserum revealed human fibronectin in some tumor cells. The cyst fluid which accumulated in the tumors contained human fibronectin as shown by a species-specific radioimmunoassay. Human fibronectin was isolated from two of the tumor fluids and compared to previously characterized fibronectins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gel electrofocusing showed that the germ-cell fibronectins resembled amniotic fluid fibronectin with regard to subunit molecular weight and isoelectric point. The distinct features of the amniotic fluid and germ-cell tumor fibronectin suggest that such fibronectins may provide new oncodevelopmental markers.

Amniotic Fluid↗
PubMed1981

Comparative studies on amniotic fluid and plasma fibronectins.

Human fibronectin was isolated from second-trimester amniotic fluid, from amniotic fluid obtained at term and from adult plasma. The amniotic-fluid fibronectins had a slightly higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than the plasma fibronectin. Early- and late-amniotic-fluid fibronectin had 9.5 and 9.6% carbohydrate respectively, whereas plasma fibronectin had 5.8%. The amniotic-fluid fibronectins had similar mannose and sialic acid contents to plasma fibronectin, but greater amounts of glucosamine, galactosamine, galactose and fucose. There were no detectable differences in the amino-acid composition of amniotic-fluid and plasma fibronectins, and the patterns of peptides obtained after tryptic digestion of fibronectin from the two sources showed extensive similarities. Fibronectins from plasma and amniotic fluid were equally active in promoting cell attachment and were immunologically indistinguishable. These results show that fibronectin from amniotic fluid is more heavily glycosylated than plasma fibronectin or previously analysed fibronectins from cultured fibroblasts. The observed differences in glycosylation may be related to cell type and/or stage of development.

Amino Acids↗
PubMed1981

Two-site sandwich enzyme immunoassay with monoclonal antibodies to human alpha-fetoprotein.

Monoclonal antibodies reacting with different antigenic determinants on the alpha-fetoprotein (AFP) molecule were used to develop a simplified 'sandwich' ELISA (enzyme-linked immunospecific assay) in which one monoclonal antibody was immobilized and another labeled with an enzyme. Due to their different specificities the antibody attached to the solid phase and the one conjugated to the enzyme did not compete for binding to AFP making it possible to complete the two antigen-antibody reactions required in a single, short incubation. The rapidity, simplicity and excellent sensitivity of this assay combined with the exquisite specificity associated with the use of two monoclonal antibodies make us believe that assays based on this principle will be useful in the quantitation of substances of medical and biological importance.

Animals↗
PubMed1981

Location of the cell-attachment site in fibronectin with monoclonal antibodies and proteolytic fragments of the molecule.

Proteolytic fragments of human plasma fibronectin were used to identify monoclonal antibodies reacting with the various domains of fibronectin. One of these antibodies, which reacts with cell-attachment-promoting fragments of fibronectin, inhibits attachment of cells to fibronectin-coated surfaces. A cell-attachment-promoting, chymotryptic, 120 kilodalton fragment was cleaved further with pepsin into three main fragments. The smallest, 15 kilodalton fragment was purified by affinity chromatography on the cell-attachment-inhibiting antibody insolubilized on Sepharose. This fragment is active in promoting cell attachment but lacks the other known binding activities of fibronectin. It can be localized between the collagen-binding and heparin-binding domains, about 127 to 197 kilodaltons from the NH2 terminus of the polypeptide. These results show that the interaction of fibronectin with cells is restricted to a defined portion of the molecule and is independent of the direct involvement of the known affinities toward other macromolecules.

Antibodies, Monoclonal↗
PubMed1981

Affinity chromatography of collagen on collagen-binding fragments of fibronectin.

The affinity of fibronectin for collagen was exploited in biospecific affinity chromatography of collagen. Matrices with excellent capacity and stability were obtained by coupling collagen-binding fragments of fibronectin to Sepharose. Collagen-binding tryptic (30,000 daltons) and chymotryptic (45,000 daltons) fragments, lacking the binding sites of intact fibronectin for various other substances, were coupled to Sepharose and used to chromatograph gelatin and type I collagen. Gelatin was rapidly and quantitatively bound to these matrices at 4 degrees C and 37 degrees C, while binding of type I collagen took place more slowly and was temperature dependent. The collagen did not bind at 4 degrees C but bound quantitatively at 37 degrees C if preincubated at this temperature. These results suggest that a temperature-dependent perturbation of the triple helical structure of the collagen uncovers the binding site for fibronectin, allowing the collagen to bind to the insolubilized collagen-binding fibronectin fragments. Enzyme affinity assays showed that the conformational change in the fibronectin-binding region of collagen was irreversible. Affinity chromatography on collagen-binding fragments of fibronectin could provide a method for the study of structure and function of collagens and may prove useful for the isolation of minor collagens.

Binding Sites↗