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PubMed1983

In vitro formation of disulfide-bonded fibronectin multimers.

Fibronectin purified from a plasma protein side fraction in the absence of denaturant contained 1.5 to 1.9 cryptic free sulfhydryl groups per 200- to 250-kDa subunit. Exposure of sulfhydryl groups in physiologic salt solutions required at least 1 M guanidine, and 3 M guanidine was required for optimal exposure. The sulfhydryl groups were not exposed by collagen, a fibronectin-binding collagen fragment, fibrinogen, heparin, hyaluronic acid, calcium ion, EDTA, deoxycholate, or methylamine. One- and two-dimensional gel electrophoresis indicated that a molecule of 40-60 kDa was disulfide-bonded to a minor portion of the fibronectin in whole human plasma and in preparations of purified fibronectin. In addition, traces of disulfide-bonded multimers were present in preparations of purified fibronectin. The proportion of fibronectin in disulfide-bonded multimers increased in guanidine-containing solutions. Compared to dimeric fibronectin, these multimers had limited solubility in physiologic buffers, could be readily cross-linked by Factor XIIIa, and exhibited altered tryptic susceptibility. In free sulfhydryl groups were blocked by prior alkylation with N-ethylmaleimide or iodoacetamide, fibronectin did not form disulfide-bonded multimers in guanidine-containing solutions. The patterns of altered tryptic susceptibility and cyanide cleavage suggested that multimer formation is mediated by both sulfhydryls of fibronectin. The transition from dimeric to multimeric fibronectin can serve as a model for the formation of disulfide-bonded fibronectin multimers in the extracellular matrix.

Disulfides↗
PubMed1981

Similarities and differences between the fibronectins of normal and transformed hamster cells.

Fibronectins from normal and virally transformed hamster cells were compared by several criteria. The fibronectin from transformed cells was similar to that from normal cells in being an intact dimeric glycoprotein with the ability to bind to gelatin, activated thiol-Sepharose, and cells. No evidence was found for proteolytic cleavages or abnormalities in disulfide bonding of transformed cell fibronectin. This fibronectin was also shown to be active in promoting cell attachment, elongation, and alignment. Therefore, the fibronectin produced by transformed cells is not defective. However, it was shown that the transformed cells were partially deficient in their capacity to bind fibronectins from either normal or transformed cells. This deficiency has implications for the significance of the loss of fibronectin on oncogenic transformation. Partial proteolysis of the fibronectins from normal and transformed cells gave rise to the same fragments. However, the glycosylated fragments from transformed cell fibronectin appeared somewhat larger than those from normal cell fibronectin. Analysis of fibronectin glycopeptides showed that transformation leads both to more branches per core and to a higher sialylation of the asparagine-linked complex carbohydrate side chains.

Animals↗
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↗
PubMed1983

Fibronectin beneath reepithelializing epidermis in vivo: sources and significance.

Fibronectin and fibrinogen occur under the migrating epidermal tongue during reepithelialization of an excisional wound, and fibronectin increases in conjunction with capillary and fibroblast ingrowth during wound healing. Although we have previously shown that fibronectin is produced by proliferating blood vessels, the source of fibronectin associated with reepithelialization and fibroblast ingrowth has not been determined. In this report we demonstrate that subepidermal fibronectin derives mostly from plasma early in reepithelialization of an excisional wound and comes from both plasma and in situ production late in reepithelialization. This finding was established by extirpating 3 mm of skin from the center of a well-healed rat xenograph on the flanks of immunosuppressed mice, harvesting the open wound sites at 2, 4, 7, and 10 days after injury, and staining the specimens with reciprocal species-specific anti-fibronectin antibodies conjugated with fluorescein. In the first 4 days after wounding, newly forming rat epidermis migrated mainly over mouse fibronectin. In contrast, by 7 days after excision, the rat epidermis transits over a matrix containing both mouse and rat fibronectin, or rat fibronectin alone, indicating that a major component of the fibronectin is produced in situ. Although the biologic significance of these observations has not been fully elucidated, fibronectin may be part of a provisional matrix that functions to support, if not actively participate in, cell recruitment to sites of inflammation or wound healing.

Animals↗
PubMed1984

Mechanism of formation of disulfide-bonded multimers of plasma fibronectin in cell layers of cultured human fibroblasts.

The free sulfhydryl groups of purified plasma fibronectin were blocked by alkylation with iodoacetamide in 3 M guanidine hydrochloride at pH 7.0 (to form fCam-fibronectin). 125I-fCam-fibronectin bound to cell layers of cultured human fibroblasts with kinetics similar to binding of 125I-labeled underivatized fibronectin. Once bound to the cell surface, 125I-fCam-fibronectin, like 125I-fibronectin, became incorporated into a detergent-insoluble extracellular matrix. Matrix-bound 125I-fCam-fibronectin, like matrix-bound 125I-fibronectin, was present as disulfide-bonded multimers as well as disulfide-bonded dimers. These results indicate that the mechanism of formation of disulfide-bonded fibronectin multimers does not involve oxidation of free sulfhydryls. Catheptic D digests of matrix-bound fibronectin were analyzed by gel electrophoresis without and with reduction. In non-reduced digests, the disulfide-rich 70-kDa amino-terminal fragment was found in a large (greater than 300 kDa) complex. Thus, fibronectin multimer formation probably occurs by disulfide exchange in the amino-terminal portion of the molecule.

Cathepsin D↗
PubMed1984

Fibronectin-induced agglutination of Staphylococcus aureus correlates with invasiveness.

Studies on the interactions of staphylococci with fibrinogen, fibrin split products, and prothrombin have formed the basis for the clumping tests for coagulase and fibrin degradation products. We investigated the role of another circulating protein, fibronectin, in clumping Staphylococcus aureus. Fibronectin is a dimeric glycoprotein with high molecular weight that is present in both blood and tissue and is involved in opsonization, clotting, healing of wounds, cell-to-cell attachment, and differentiation. Each fibronectin molecule has two S. aureus binding sites, thus allowing lattice formation. We defined conditions under which fibronectin will cause agglutination of S. aureus. Strains of S. aureus that were most easily clumped had the largest number of fibronectin receptors. Trypsinization or gentle sonication removed the fibronectin binding and agglutinating receptors from S. aureus. These treatments did not alter viability, which suggests that binding is a superficial component of the organisms. Invasive fibronectin-binding strains were from a wide variety of phage types. Twenty two S. aureus isolates from patients with invasive disease were more readily agglutinated and had a greater number of fibronectin binding sites than 19 noninvasive strains (p less than 2.5 X 10(-4)). This suggests that the pathogenicity of S. aureus invasion may be enhanced by binding of bacteria to tissue fibronectin or by agglutination of bacteria by plasma fibronectin. Thus, the fibronectin receptors on S. aureus that mediate agglutination might also permit invasion of host tissues.

Fibrinogen↗
PubMed1984

Multiple fibronectin subunits and their post-translational modifications.

We report analyses of fibronectin subunit diversity by high resolution one- and two-dimensional gel electrophoresis. We have studied plasma and cellular fibronectins of rats and hamsters. Each form of fibronectin comprises multiple distinguishable subunits and, within each rodent species, all subunits of plasma fibronectin are resolvable from those of cellular fibronectin. Some, but not all, of this heterogeneity is caused by differential glycosylation. Thus, while glycosylated plasma and cellular fibronectins share no common subunits, nonglycosylated forms of these proteins appear to share 2-3 subunits. In addition, there are subunits unique to plasma and to cellular fibronectins in both rats and hamsters, although the pattern of diversity differs slightly between species. All size variants of fibronectin are phosphorylated to varying degrees. However, only some subunits are sulfated, apparently on tyrosine residues in the C-terminal third of the molecule. Comparison of the distribution of sulfate on the various fibronectin subunits with recent results on generation of multiple mRNAs by alternative splicing suggests that tyrosine sulfate is located in a polypeptide segment present in only certain fibronectin subunits. The results reported here provide information on the likely contributions of primary sequence differences and post-translational modifications to the heterogeneity of fibronectin subunits.

Animals↗
PubMed1984

Binding of fibronectin by the acute phase reactant C-reactive protein.

Following tissue injury, the concentration of C-reactive protein (CRP) is known to increase in plasma rapidly, while that of fibronectin often decreases. We now report that CRP immobilized onto polystyrene surfaces binds soluble plasma fibronectin (Kd = 1.5 X 10(-8) M). The binding of fibronectin by CRP was relatively sensitive to ionic conditions, being maximal at physiological NaCl concentrations. A decrease of pH from neutral to 5-6 greatly enhanced the binding of fibronectin by CRP. Ca2+ ions at greater than 1 mM inhibited binding. No binding was observed between fibronectin and CRP in soluble phase. CRP was found also to bind fibrinogen, which competed with fibronectin for CRP-binding sites. This was shown to explain why fibronectin was effectively bound from serum but not from plasma by immobilized CRP. The amount of CRP immobilized was critical in binding fibronectin; a too dense molecular layer of CRP inhibited the binding, as did the postsaturation of free surfaces with albumin, which itself was not bound by CRP. Soluble fibronectin agglutinated CRP-coated latex particles. Most or all of the CRP-binding activity in the fibronectin molecule was localized to the 120-140-kilodalton fragment, which also contains cell-binding and heparin-binding domains of fibronectin. The results provide a link between acute phase response and tissue repair.

C-Reactive Protein↗
PubMed1980

Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments.

We have identified the biological activity of three polypeptides released by limited proteolysis of human plasma fibronectin by leukocyte elastase. A Mr = 140,000 peptide contains cell-spreading activity; a Mr = 60,000 peptide mediates binding to denatured collagen (gelatin), and a Mr = 29,000 peptide contains glutaminyl residues responsible for the transglutaminase (blood coagulation factor XIIIa)-catalyzed incorporation of amines. More extensive proteolysis yielded numerous peptides, including a Mr = 40,000 peptide derived from the Mr = 60,000 peptide which retains gelatin-binding activity. Quantification of the gelatin-binding peptides is consistent with two binding sites per dimeric fibronectin molecule of Mr = 440,000. Both Mr = 60,000 and 40,000 gelatin-binding peptides were enriched with half-cystine residues, containing 28 and 25, respectively, but devoid of cysteine. This, coupled with the electrophoretic behavior of both peptides, was consistent with the presence of intramolecular disulfide bonds in the gelatin-binding domain. Intact fibronectin contains 1 free cysteine residue/monomer, as recently described. This cysteine reacts with 5,5'-dithiobis(2-nitrobenzoic acid) very slowly under nondenaturing conditions but rapidly when fibronectin is denatured. The free cysteine is located in the Mr = 140,000 peptide. While the Mr = 40,000 and 60,000 gelatin-binding peptides bind to gelatin with an affinity about 30-fold and 5-fold less than intact fibronectin (based on a monomeric fibronectin Mr = 220,000), neither gelatin-binding peptide supports spreading of fibronectin-deficient test cells on gelatin or tissue culture plastic substrates. The purified Mr = 140,000 peptide supported cell spreading on plastic, retaining about one-half of the spreading activity of intact fibronectin on a weight basis. These data confirm recent results, suggesting multiple, protease- resistant domains with discrete biological functions within fibronectin. Our results, together with established data, suggest a model for the location of the transglutaminase-reactive glutaminyl residues, gelatin binding, and cell-adhesive domains in fibronectin. The release of univalent, biologically active fibronectin fragments by elastase, a major physiologically released inflammatory protease of human leukocytes, suggests a new potential mechanism for alteration of cell connective tissue interactions at sites of inflammation in vivo.

Animals↗
PubMed1981

Fibronectin production by human mammary cells.

Human mammary cells were examined for the presence of the high-molecular-weight surface glycoprotein fibronectin. Early passage mammary epithelial cell and fibroblast cultures from both carcinomas and normal tissues were tested for the presence of cell-associated fibronectin by immunofluorescence microscopy and for the synthesis and secretion of fibronectin by specific immunoprecipitation of metabolically labeled protein. In vivo frozen sections of primary carcinomas and normal tissues were tested for the localization of fibronectin by immunofluorescence microscopy. In contrast to the extensive fibrillar networks of fibronectin found in the fibroblast cultures, the epithelial cell cultures from both tissue sources displayed a pattern of cell-associated fibronectin characterized by powdery, punctate staining. However, the cultured epithelial cells, as well as the fibroblasts, secreted large quantities of fibronectin into the medium. Putative myoepithelial cells also displayed extensive fibrillar networks of fibronectin. The difference in cell-associated fibronectin distribution between the epithelial cells and the fibroblasts and putative myoepithelial cells provided a simple means of quantitating stromal and myoepithelial cell contamination of the mammary epithelial cells in culture. In vivo, normal tissues showed fibronectin primarily localized in the basement membrane surrounding the epithelial cells and in the stroma. Most primary carcinomas displayed powdery, punctate staining on the epithelial cells in addition to the fibronectin present in the surrounding stroma.

Basement Membrane↗
PubMed1981

Fibronectin in healing rabbit corneal wounds.

Fibronectin, a glycoprotein present in plasma and extracellular matrix, is believed to be involved in cell-cell and cell-matrix interactions. Using immunofluorescence techniques, we studied the time course of appearance and distribution of fibronectin in healing rabbit corneal epithelial wounds and compared fibronectin to other selected proteins in the cornea. In the normal cornea, fibronectin was detected only in Descemet's membrane and not in the epithelial basement membrane. Shortly after wounding, fibronectin deposited on the denuded corneal surface and was a continuous prominent layer by 8 hours. The epithelium had begun to migrate over the deposited fibronectin by 22 hours and by 52 hours had completely covered the denuded surface. Fibrinogen/fibrin was also detected on the initial bare wound surface. Once the wound was reepithelialized, the subepithelial fibronectin and fibrin layer then progressively disappeared, so that by 2 weeks only a small amount was detected. Fibronectin also appeared in the deep stroma of corneas after wounding in elongated patches, a pattern suggestive of keratocyte association. Fibronectin had an inverse relationship to bullous pemphigoid antigen which was used as a marker for the lamina lucida of the epithelial basement membrane. The bullous pemphigoid antigen, which was found in the normal corneal epithelial basement membrane, was removed with the epithelium and reappeared during the wound healing when fibronectin was diminishing. IgG and albumin did not localize on the wound surface, and the diffuse staining seen in the stroma did not change during healing. These findings are compatible with the hypothesis that fibronectin and fibrin play a role in epithelial migration and temporary adhesion to the surface during corneal wound healing, at a time when the normal anchoring mechanism is lost.

Albumins↗
PubMed1982

Conformational states of fibronectin. Effects of pH, ionic strength, and collagen binding.

Human plasma fibronectin was enzymatically labeled with dansylcadaverine using plasma Factor XIIa. Fluorescence polarization studies of dansylcadaverine-labeled fibronectin indicate that fibronectin has a significant degree of chain flexibility in physiologic solution and that there is an increase in chain flexibility at high pH or ionic strength. Binding of a collagen peptide to dansylcadaverine-fibronectin results in a decrease in fluorescence polarization, suggesting that such binding causes a conformational change which also results in increased chain flexibility.l Quasielastic light scattering and intrinsic viscosity measurements of fibronectin were performed under physiologic conditions and at high pH and ionic strength. Shape calculations based on these data indicate that fibronectin is in an elongated configuration under physiologic conditions and further unfolds at high pH or ionic strength into a very flexible, strand-like configuration. Light scattering studies of fibronectin after binding of a collagen fragment indicate that such binding results in a decrease in the diffusion coefficient, suggesting that collagen binding also results in a partial unfolding of fibronectin. These results suggest that published electron micrographs of fibronectin showing a long, strand-like molecule do not reflect the conformation of plasma fibronectin under physiologic conditions; fibronectin, however, may assume an unfolded conformation upon binding to collagen in the tissue matrix.

Cadaverine↗
PubMed1981

Humoral regulation of plasma opsonic fibronectin levels: its role in reticuloendothelial recovery after RE blockade.

The present study in rats evaluated the concept that a humoral factor regulates opsonic fibronectin levels. Intravenous administration of gelatin-coated particles depletes immunoreactive opsonic fibronectin by 95% within 30 minutes. Partial restoration of opsonic protein was observed by 3 hours with complete recovery 6-24 hours. Post-blockade serum was then evaluated with respect to this humoral factor. Controls were given either normal serum or serum rendered fibronectin-deficient by affinity chromatography. Affinity-adsorbed fibronectin-deficient serum did not increase immunoreactive opsonic fibronectin. Injection of normal serum resulted in the expected elevation in fibronectin due to its fibronectin content. In contrast, injection of fibronectin-deficient serum obtained during RE blockade dramatically elevated (P less than 0.02) immunoreactive opsonic fibronectin to 160.6 +/- 14% of control levels by 3 hours. Thereafter, fibronectin levels returned to control over the 6-24 hour period. Our temporal studies suggest that a circulating transferable factor, which may be a hormone, may influence the synthesis and/or release of opsonic fibronectin.

Animals↗
PubMedDate not supplied

Immunohistochemical detection of fibronectin in early and advanced atherosclerosis.

Fibronectin is known to be involved in the pathogenesis of atherosclerosis. We therefore applied paraffin-section immunohistochemistry, which permits detailed morphological analysis of the tissue distribution of fibronectin, in order to evaluate the alterations of fibronectin in terms of quantity and localization during the progression of atherosclerosis in humans. In 48/60 (80%) cases (p < 0.005) with early atherosclerotic lesions we found intense fibronectin staining presenting a distribution in continuous fibrils in the subendothelium around the fibrous plaque. The total amount of fibronectin was elevated. In 51/60 (85%) cases (p < 0.001) presenting advanced or complicated atherosclerotic lesions, the characteristic distribution of fibronectin in continuous fibrils was interrupted, fibronectin strands were fragmented and in regions where the sclerotic process was complete, fibronectin seemed to be almost absent. These findings reveal that fibronectin during atherosclerosis plays a role similar to that reported during the wound healing process and demonstrate a close relationship between quantity and topographical distribution of fibronectin and evolution of atherosclerosis. Further studies are required to determine whether fibronectin could be used as a marker for evaluation of the severity of atherosclerotic lesions.

Adult↗
PubMed1994

The collagen binding domain of fibronectin contains a high affinity binding site for Candida albicans.

A 30-kDa proteolytic fragment from the gelatin/collagen-binding domain of fibronectin is a potent inhibitor of fibronectin binding to Candida albicans, with a molar inhibition constant equal to that of intact fibronectin. Recombinant and proteolytic fragments from the cell-, the fibrin I-, and the heparin II-binding domains also inhibit fibronectin binding, but are 13-1000-fold less active. In suspension, binding of fibronectin to C. albicans is regulated by growth conditions and is specific, saturable, time-dependent, reversible, and divalent cation-independent. Scatchard plot analyses indicate the presence of high affinity (Kd = 1.3 x 10(-9) M) and low affinity (Kd = 1.2 x 10(-7) M) receptors. Recombinant or proteolytic fragments from four binding domains of fibronectin promote adhesion of C. albicans. A recombinant fragment corresponding to the cell-binding domain but with the sequence Arg-Gly-Asp-Ser deleted promotes C. albicans adhesion and inhibits fibronectin binding to C. albicans with the same activity as the natural sequence. Furthermore, four peptides containing the Arg-Gly-Asp-Val sequence and the peptides CS-1 and Arg-Glu-Asp-Val did not block the binding of fibronectin to C. albicans. Thus, in contrast to the specific binding of soluble fibronectin, recognition of immobilized fibronectin by C. albicans is mediated by several domains of the protein. Interactions with the cell-binding domain are not mediated by the Arg-Gly-Asp or other known recognition sequences as it has been suggested. Binding of fibronectin also did not correlate with C3d binding to the avirulent clones of C. albicans strain H12 or with iC3b binding to variants of the strain 4918.

Amino Acid Sequence↗
PubMed1996

Glomerular basement membrane-derived perlecan inhibits mesangial cell adhesion to fibronectin.

To explore whether the heparan sulfate proteoglycan, periecan, may act as a regulator of glomerular cell behavior, we examined the effects of perlecan on adhesive properties of rat and human mesangial cells in culture. We observed that mesangial cells did not adhere to purified perlecan. Mesangial cell adhesion to fibronectin, but not to laminin, was inhibited when perlecan was present in the substratum, reaching complete inhibition at 20 micrograms/ml perlecan. Similarly, perlecan reduced mesangial cell adhesion to the 120 kDa fragment of fibronectin and to bovine serum albumin-coupled RGD peptides both lacking the fibronectin heparin-binding domains, indicating that the inhibitory effect of perlecan does not require interaction of its heparan sulfate chains with fibronectin. The core protein of perlecan seems to be relevant, since perlecan retained most of its anti-adhesive potency after total deglycosylation. It is unlikely that perlecan causes steric inhibition of the cell binding sites of fibronectin by direct binding, since the 120 kDa fragment of fibronectin which includes the cell binding site, did not bind to intact perlecan. In the presence of submaximal inhibitory concentrations of perlecan, soluble RGD peptides (100 micrograms/ml), by themselves without effect, completely inhibited cell adhesion to fibronectin. Using immunocytochemistry, we examined the effects of perlecan on the organization of fibronectin-specific, RGD-dependent beta 1 integrins in focal contacts. When perlecan was added to the substratum, human mesangial cells adhered to fibronectin only in the presence of collagen I. Under these conditions, perlecan did not significantly alter the organization of alpha 5 beta 1 integrin into focal contacts of mesangial cells which adhered to a substratum composed of collagen I plus fibronectin. These data suggest that perlecan exerts its anti-adhesive effects on mesangial cells via a core protein-dependent mechanism which reduces the avidity of the fibronectin receptor. The resulting avidity level is too low for mediating cell binding but sufficient to induce integrin organization into focal contacts.

Animals↗
PubMed1997

Loss of EDB+ fibronectin isoform is associated with differentiation of alveolar epithelial cells in human fetal lung.

Cell-matrix interactions have been shown to regulate the development of the lung, particularly airway branching and alveolarization. Fibronectin is the major constituent of pulmonary extracellular matrix and exists in multiple isoforms arising from alternative RNA splicing. EDA and EDB are the two major alternatively spliced segments, the expression of which is regulated in a spatiotemporal and oncodevelopmental manner. In this study, we investigated immunohistochemically the distribution of the EDA- and EDB-containing fibronectin isoforms (referred to as EDA+ fibronectin and EDB+ fibronectin, respectively) in normal and hypoplastic human lungs at different gestational ages to explore the role of these fibronectin isoforms in alveolarization. EDA+ fibronectin was expressed around the distal airspaces throughout the development of both normal and hypoplastic lungs. In contrast, the expression of EDB+ fibronectin was restricted to the lung with morphologically immature acinar complex, typically observed in normally developing lungs of < 30 gestational weeks or in hypoplastic lungs. To further confirm the restricted expression of EDB+ fibronectin in immature acinar complex, we examined the correlation of EDB+ fibronectin expression with that of the surfactant protein SP-A, a biochemical marker for the differentiated type II pneumocytes. A clear inverse relationship between the immunoreactivities for EDB+ fibronectin and SP-A was observed in both control and hypoplastic lungs. Given the proposed importance of fibronectins in the differentiation of alveolar epithelial cells, our results suggest that the EDB segment plays a regulatory role in the differentiation of immature acinar epithelial cells into type II pneumocytes. The EDB segment may also serve as a new histochemical marker for the functional maturity of fetal lung tissues.

Alternative Splicing↗
PubMed1979

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.

Animals↗