Quantification Of Fibronectin Research Articles

Quantification of Fibronectin 1 (FN1) splice variants, including two novel ones, and analysis of integrins as candidate FN1 receptors in bovine preimplantation embryos

BackgroundFibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development.ResultsRT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells.In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 α integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 β integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV).ConclusionThe existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.

Quantification of fibronectin and cell surface interactions by AFM

The cell adhesion strength between FN and the living cell was successfully examined using an AFM cantilever having a FN-coated spherical microbead as a probe. The microbead was used to increase (by some 55-fold) the area of contact with the cell surface. Fibronectin (FN) was chosen as the ligand and immobilized on the bead. The area under the force-extension curve obtained during retraction of the FN-coated tip from the cell surface after contact was defined as ‘separation work’ and used as a parameter to evaluate the strength of the interaction. In previous studies, final rupture forces were taken to represent the strength of interactions, but in the present study using a large contact area, separation work was expected to reflect the overall adhesion strength in the contact area. The mean separation work between a FN-coated bead and mouse fibroblast cell was 0.69±0.45×10 −17 J/μm 2. In the presence of 0.1 mg/ml FN in the scanning solution, this value decreased to 0.10±0.08×10 −17 J/μm 2. The separation work between an unmodified microbead and cell was calculated to be 0.15±0.13×10 −17 J/μm 2. Mapping of the separation work over a limited area of the cell surface was found to be reproducible for six repeated scans and the distribution of FN-binding sites on the cell surface was investigated by mapping over a large area. The results showed that separation work is a good measure of specific interactions between FN and the cell surface, and the system developed here is a promising quantitative method to evaluate cell adhesion force.

Quantification of Fibronectin in Bronchoalveolar Lavage Fluid and in Lung Parenchyma: Differences between Smokers and Non-Smokers

Fibronectin (FN) is a glycoprotein present in the epithelial surface of airways and in the interstitial parenchyma of the lung. The aim of this study was to quantify the amount of FN in human lung parenchyma, and to investigate possible differences between smokers and non-smokers, both in lung parenchyma and in bronchoalveolar lavage (BAL) fluid. BAL fluid was recovered in 21 individuals. Quantification of FN in BAL was done by a non-competitive ELISA. Twenty-six lung parenchyma samples were also obtained from lung resections. FN was extracted with urea and heparin. Quantification of FN in lung parenchyma was determined by a competitive ELISA. The mean +/- SD FN content in BAL fluid from smokers was 1.59 +/- 1.65 micrograms/mg total protein and 0.53 +/- 0.26 in non-smokers (p < 0.05). However, the amount of FN/ml BAL fluid was not statistically different between smokers (0.15 +/- 0.13 micrograms/ml) and non-smokers (0.106 +/- 0.07). The mean extractable FN in human lung tissue was 38.6 micrograms/g wet weight (31.7-45.5, 95% mean confidence interval) with no statistical difference between smokers and non-smokers. We conclude that in smokers, FN accounts for a higher proportion of the total protein in BAL fluid, and that there were no differences in FN content in lung parenchyma between smokers and non-smokers.

Quantification of fibronectin in extracts of human aortae by an ELISA

Quantification of fibronectin in extracts of human aortae by an ELISA

Quantification of fibronectin in extracts of human aortae by an ELISA

A procedure for extraction and quantification of fibronectin in human aortic tissue is described in this paper. Dried, defatted samples of human aortic tissue were subjected to sequential extraction with (i) 0.89% NaCl, 10 mmol/l Tris/HCl, pH 7.4, (ii) 5 mg/ml heparin, 2 mol/l urea and (iii) collagenase digestion. More than 75% of hexosamine-containing molecules were solubilized by this procedure. Immunoblotting of extracted proteins separated by SDS-PAGE showed that extracted fibronectin had a mobility in the same range as that of plasma fibronectin. Fibronectin ELISA performed on these extracts gave dilution curves parallel to the standard curve, the sensitivity was 2.7 micrograms/l. Recoveries of a fibronectin standard added to the NaCl, heparin/urea and collagenase solutions during extraction were 97%, 90% and 84% respectively. Normal aortic tissue from 31 patients was subjected to the sequential extraction scheme and fibronectin quantification in the various extracts demonstrated that 4.52 +/- 1.79 micrograms was dissolved in the NaCl extracts, 5.41 +/- 2.28 in the heparin/urea extract and 1.08 +/- 0.43 in the collagenase digest, respectively. (Values are expressed as micrograms fibronectin/10 mg dry, defatted tissue (mean +/- SD]. Our results indicate that the ELISA method can be applied for the measurement of fibronectin in extracts of human aortic tissue. This might be useful in the study of diseases where alterations in arterial fibronectin content may be expected.

Studies on cellular adhesiveness in five different culture cell lines derived from carcinoma of the urinary bladder.

Study on biological behaviors of human carcinoma cells with special em-phasis placed on intercellular and cell-to-matrix adhesiveness of cultured cells was undertaken. Characterization of five different cell lines derived from bladder carcinoma was morphologically determined utilizing monolayer, rota-tion and collagen sponge matrix culture. Also determined were distribution and quantification of fibronectin in cells and cultured media by immuno-fluorescence technique and immunoelectrophoresis.The results showed that regardless of the culture technique employed, distinct morphological characteristics of cellular arrangement and surface of the cells, which well corresponded to the grade of the original carcinoma of the bladder. The adhesiveness tended to be inversely proportional to grade of the original carcinoma, however, KU-7 derived from low grade carcinoma was shown to have decreased adhesiveness and increased amount of fibronectin in its culture media compared with other four cell lines.These results indicate a close association of adhesiveness of cultured cells with cellular malignancy and its proliferation. Studies of tumor cell adhesive-ness may thus provide insight into the mechanism of local tumor growth and dissemination to form distant metastasis.