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B Carnemolla

Publications and source records attributed to B Carnemolla.

At least 37 records · Page 2Linked to original sources
PubMed1989

A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors.

Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla, A. Siri, T. E. Petersen, G. Paolella, G. Sebastio, and F. E. Baralle. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:2337-2342). Here we report on the production and characterization of a monoclonal antibody (BC-1) which recognizes an epitope within the protein sequence coded for by the ED-B exon. This monoclonal antibody makes it possible to carry out immunohistochemical analysis of the distribution of the ED-B-containing FN isoform (B-FN) in human tissues. The results show that while in normal, adult, human tissues total FN has a widespread distribution, the B-FN isoform is restricted only to synovial cells, to some vessels and areas of the interstitium of the ovary, and to the myometrium. On the contrary, the B-FN isoform has a much greater expression in fetal and tumor tissues. These results demonstrate that, in vivo, different FN isoforms have a differential distribution and indicate that the B-FN isoform may play a role in ontogenesis and oncogenetic processes.

Antibodies, Monoclonal↗
PubMed1988

Modulation of matrix adhesive responses of human neuroblastoma cells by neighboring sequences in the fibronectins.

Attachment and neurite extension have been measured when Platt or La-N1 human neuroblastoma cells respond to tissue culture substrata coated with a panel of complementary fragments from the individual chains of human plasma (pFN) or cellular fibronectins (cFN) purified from thermolysin digests. A 110-kD fragment (f110), which contains the Arg-Gly-Asp-Ser sequence (RGDS)-dependent cell-binding domain but no heparin-binding domains and whose sequences are shared in common by both the alpha- and beta-subunits of pFN, facilitated attachment of cells that approached the level observed with either intact pFN or the heparan sulfate-binding platelet factor-4 (PF4). This attachment on f110 was resistant to RGDS-containing peptide in the medium. Neurite outgrowth was also maximal on f110, and half of these neurites were also resistant to soluble RGDS peptide. Treatment of cells with glycosaminoglycan lyases failed to alter these responses on f110. Therefore, there is a second "cell-binding" domain in the sequences represented by f110 that is not RGDS- or heparan sulfate-dependent and that facilitates stable attachment and some neurite outgrowth; this domain appears to be conformation-dependent. Comparisons were also made between two larger fragments generated from the two subunits of pFN-f145 from the alpha-subunit and f155 from the beta-subunit--both of which contain the RGDS-dependent cell-binding domain and the COOH-terminal heparin-binding domain but which differ in the former's containing some IIICS sequence at its COOH terminus and the latter's having an additional type III homology unit. Heparin-binding fragments (with no RGDS activity) of f29 and f38, derived from f145 or f155 of pFN, respectively, and having the same differences in sequence, were also compared with f44 + 47 having the "extra domain" characteristic of cFN. Attachment on f145 was slightly sensitive to soluble RGDS peptide; attachment on f155 was much more sensitive. There were also differences in the percentage of cells with neurites on f145 vs. f155 but neurites on either fragment were completely sensitive to RGDS peptide. Mixing of f29, f38, or PF4 with f110 could not reconstitute the activities demonstrated in f145 or f155, demonstrating that covalently linked sequences are critical in modulating these responses. However, mixing of f44 + 47 from cFN with f110 from pFN increased the sensitivity to RGDS peptide.(ABSTRACT TRUNCATED AT 400 WORDS)

Axons↗
PubMed1987

Localization of the cellular-fibronectin-specific epitope recognized by the monoclonal antibody IST-9 using fusion proteins expressed in E. coli.

Here we report on a monoclonal antibody (IST-9) which distinguishes between human cellular and plasma fibronectin. Using beta-galactosidase-fibronectin fusion proteins expressed in E. coli we have demonstrated that this monoclonal antibody is specific for a fibronectin segment (ED) which can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Furthermore, using the same fusion proteins we have been able to localize precisely the epitopes of two other monoclonal antibodies (IST-1 and IST-2), specific for the heparin-binding domain 5 of fibronectin.

Antibodies, Monoclonal↗
PubMed1987

Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon.

Purification and amino acid sequence analysis of a proteolytic fragment of fibronectin (FN) from transformed human cells demonstrated that a high percentage of these FN molecules contains an extra amino acid sequence which is present only in a very low percentage of FN molecules from normal fibroblasts and is undetectable in plasma FN. This new amino acid sequence introduces into the FN molecule a site very sensitive to a number of proteolytic enzymes. By analyzing the cellular mRNA and genomic clones, we have demonstrated that this sequence derives from a differential splicing pattern of the FN mRNA precursors, which leads in transformed cells to a high-level expression of an extra type III homology repeat (ED-B) coded for by a previously unobserved exon. Here we also report the complete sequence of this new exon. These results demonstrate that in malignant cells the mechanisms regulating the splicing of FN mRNA precursors are altered.

Amino Acid Sequence↗
PubMed1987

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells.

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.

Amino Acid Sequence↗
PubMed1986

DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin.

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.

Amino Acid Sequence↗
PubMed1985

Elution of fibronectin proteolytic fragments from a hydroxyapatite chromatography column. A simple procedure for the purification of fibronectin domains.

Human plasma fibronectin is composed of at least five distinct domains which we refer to as Hep-1/Fib-1, Gel, Cell, Hep-2 and Fib-2 depending on their affinity for heparin (Hep), gelatin (Gel), the cell surface (Cell) or fibrin (Fib). These domains are aligned from the NH2 to the COOH terminus in the above order and can be separated from each other by mild proteolytic digestion. We have studied the elution of fibronectin thermolysin digest from a hydroxyapatite column using a linear gradient (0.5-190 mM) of sodium phosphate buffer. The five major fibronectin domains were eluted from the hydroxyapatite chromatography column in the following order: Gel, Fib-2, Cell, Hep-1/Fib-1 and Hep-2. They were identified on the basis of their molecular mass, affinity to different macromolecules and reaction with domain-specific monoclonal antibodies. All domains except the Cell and Hep-2 domains eluted as single homogeneous peaks. The Cell domain eluted as two different peaks and the Hep-2 domain eluted as four different peaks. This is the first time that heterogeneity of these two domains has been observed. Since chromatography of a fibronectin thermolysin digest on a hydroxyapatite column provides a good separation of the five major fibronectin domains, we have elaborated a procedure in which each fibronectin domain is purified by no more than two steps; hydroxyapatite and molecular exclusion chromatography. Fractionation of fibronectin proteolytic digest on a hydroxyapatite chromatography column should be of great value in the comparative analysis of fibronectin from different sources and in the study of fibronectin heterogeneity. Its use in combination with molecular exclusion chromatography offers a simple and high-yield method for the purification of large amounts of fibronectin domains.

Chemical Phenomena↗
PubMed1984

Characterization of synovial fluid fibronectin from patients with rheumatic inflammatory diseases and healthy subjects.

Synovial fluid fibronectin from normal subjects and from patients who have rheumatic inflammatory diseases has been studied and compared with plasma fibronectin. The average fibronectin concentration in synovial fluids from normal subjects was 172 +/- 69 micrograms/ml; it was 721 +/- 315 and 556 +/- 349 micrograms/ml in synovial fluids from patients with rheumatoid arthritis and osteoarthritis, respectively. This is the first report on fibronectin concentrations in normal synovial fluids. Synovial fluid fibronectin from healthy subjects and patients with rheumatoid arthritis or osteoarthritis showed a molecular weight identical to that of plasma fibronectin. All normal and pathologic synovial fluid fibronectins showed a remarkably lower electrophoretic mobility compared with that of plasma fibronectin, when separated according to net molecular charge on agarose gel. Peptides from thermolysin digests of fibronectin from plasma and synovial fluid, when compared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed distinct differences. These data demonstrate that synovial fluid fibronectin represents a molecular form which is structurally different from that of plasma fibronectin. This suggests that synovial fluid fibronectin is locally synthesized, possibly by a cell type which differs from that responsible for the production of the plasmatic fibronectin pool.

Aged↗
PubMed1984

Fibronectin concentrations in pleural effusions of patients with malignant and non-malignant diseases.

We have determined the amount of fibronectin in pleural fluids and plasma of 131 patients suffering from pleurisy of malignant and non-malignant etiology. We observed a significantly higher concentration of fibronectin in pleural fluids of a few patients with mesothelioma as compared with that found in pleural fluids of patients with pleurisies of different etiology. In these patients the pleural fluid fibronectin concentration was higher than that measured in plasma, suggesting local synthesis of fibronectin.

Adult↗
PubMed1983

Increased plasma fibronectin concentrations in tumor bearing mice.

Fibronectin concentration was studied in plasma of mice at different times after inoculation of 3 different experimental tumors, Colon 26 adenocarcinoma, mFS6 fibrosarcoma and B16 melanoma. A significant increase in fibronectin concentrations 10-30 days after inoculation of all tumors was found. Highest and earliest fibronectin increases were observed in BALB/c mice inoculated with Colon 26 adenocarcinoma cells. Control animals inoculated with previously irradiated cells (5000 rad) showed neither tumor growth nor significant variations in plasma fibronectin levels.

Adenocarcinoma↗
PubMed1982

Species-specific monoclonal antibodies in the assignment of the gene for human fibronectin to chromosome 2.

Using three different species-specific monoclonal antibodies we have studied, in human-mouse and human-hamster somatic cell hybrids, the correlation between the presence of different human chromosomes and the ability to release human fibronectin into the tissue culture medium. Presence of human fibronectin was determined by an affinity-radioimmunoassay. In addition, tissue culture media of the different hybrids were separated on SDS-polyacrylamide gels, the proteins were blotted onto a nitrocellulose sheet and human fibronectin visualized by an immunoenzymatic technique. Karyology and determination of isoenzyme markers of specific human chromosomes show that the ability to produce human fibronectin segregated with the presence of human chromosome 2.

Animals↗
PubMed1980

Somatic cell hybrids producing antibodies specific to human fibronectin.

We have produced somatic cell hybrids between mouse plasocytoma cells deficient in hypoxanthine phosphoribosyltransferase (P3 x 63 Ag8) and spleen cells derived from mice immunized with purified human plasma fibronectin. We report herein the properties of monoclonal anti-fibronectin antibodies released by eight different clones.

Animals↗
PubMed1980

A simplified procedure for the preparation of antibodies to serum fibronectin.

In the present paper we describe in detail a simple procedure for the preparation of monospecific antisera to human and mouse serum fibronectin. A similar procedure could also be used to prepare antibodies to fibronectin from other species. The procedure, based on the recently reported affinity of fibronectin for gelatin, essentially consists of two steps (1) Immunization of rabbits with fibronectin purified from serum by affinity chromatography using gelatin coupled to CNBr-activated Sepharose 4B. (2) Absorption of the antiserum obtained by an immunoabsorbent prepared using fibronectin-free serum proteins that remained after absorbing serum with gelatin-Sepharose. The antisera obtained were monospecific, as determined by immunoelectrophoresis and did not show any difference with respect to antisera prepared by different procedures.

Animals↗
PubMed1979

Fibronectin: a chromatin-associated protein?

We have previously reported that chromatin preparations from human cultured fibroblasts contain a single homologous serum protein. In this paper we present evidence, based on immunological identity and physicochemical properties, that this serum protein is fibronectin. Furthermore, using a radioimmunoassay system, we have estimated that fibronectin represents about 0.7% of the total protein in both chromatin preparations and whole fibroblasts. Using a nitrocellulose filter assay system, we also show that fibronectin is a DNA-binding protein having an equilibrium constant of 4.6 x 10(-6) M. Equilibrium competition experiments have demonstrated that fibronectin has the ability to differentiate among nucleotides, indicating that fibronectin-DNA interaction is at least partially specific, and that a minimum polymer length of 12-18 nucleotides is required for effective binding to occur. Fibronectin has been isolated readily from plasma using DNA-affinity chromatography. We do not have direct evidence that fibronectin is an actual nonhistone chromosomal protein, but fibronectin is a DNA-binding protein (at least under in vitro assay conditions) and appears to be a normal constituent of chromatin as chromatin is currently isolated from cell nuclei.

Antibody Specificity↗
PubMed1979

Concentration of fibronectin in plasma of tumor-bearing mice and synthesis by Ehrlich ascites tumor cells.

In the present paper we have studied: (a) the concentration of fibronectin (FN) in plasma and in ascitic fluid of mice at different times after inoculation of Ehrlich ascites tumor cells; (b) the ability of Ehrlich ascites cells to synthesize and release FN; and (c) the localization of FN in Ehrlich ascites cells by immunofluorescence microscopy. It was found that (a) 4 to 5 days after inoculation of the tumor, the plasma concentration of FN was significantly higher [1.7 +/- 0.07% (S.E.) of total plasma protein] than that in the normal control mice (0.8 +/- 0.035); (b) FN is present in the ascitic fluid in all phases of tumor growth; (c) Ehrlich ascites cells cultured in vitro synthesize and release large amounts of FN in the culture medium; and (d) only about 1 to 2% of the tumor cells show a very small amount of FN, and this is mostly in the area of cell-cell contact.

Animals↗