[Current status in gynecological infectious diseases and the possibilities of cure].
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Female↗
Biomedical subjects
M Vuento
Publications and source records attributed to M Vuento.
Solution structure of human plasma fibronectin using small-angle X-ray and neutron scattering at physiological pH and ionic strength.
Human plasma fibronectin has been investigated at physiological pH and ionic strength, by using small-angle X-ray and neutron scattering techniques. The results indicate that the molecule is disc shaped with an axial ratio of about 1:10. In fact, an ellipsoid of revolution with semiaxes a = 1.44 nm and b = c = 13.8 nm is in agreement with the experimental scattering data, and can also fully explain the rather extreme hydrodynamic parameters reported for fibronectin. The X-ray data gave a radius of gyration of 8.9 nm and a molecular weight of 510,000, whereas the neutron data gave slightly larger values, 9.5 nm and 530,000, respectively. From the volume of the best fitting ellipsoid we obtain a degree of hydration of 0.61 g H2O/g protein (dry weight). Neutron data, recorded at different D2O concentrations in the solvent, gave a match point of 43% D2O, which indicates that approximately 80% of the hydrogens bound to oxygen and nitrogen are exchangeable.
Fibronectins↗
Fibronectin fragmentation induced by dental plaque and Bacteroides gingivalis.
Degradation of fibronectin (FN) by subgingival and supragingival plaque and Bacteroides gingivalis (Bg) was studied in vitro. The degradation of FN by both types of plaque was relatively rapid, continuous but incomplete. Some differences were found between supra- and subgingival samples. Supragingival plaque extracts produced several FN fragments of 110-180 kd during short incubations of 15-60 min. The predominant fragment after overnight incubation was a 110 kd polypeptide. With subgingival plaque extract a more extensive degradation of FN was noted. The main degradation product was a 120 kd fragment after overnight incubation. Several peptide fragments were released from fibronectin by Bg extracts. Their molecular size was different from those produced by trypsin, elastase or dental plaque. When cell extracts of Bg were fractionated by high performance liquid chromatography, three separate peaks of fibronectin degrading activity were obtained. Two of those peaks also contained trypsin-like enzyme activity. The degradation of fibronectin and the subsequent formation of biologically active peptides may have many effects in periodontal pockets. These may include modifying effects on plaque growth and wound healing.
Bacteroides↗
Secondary structure of human plasma fibronectin: conformational change induced by calf alveolar heparan sulfates.
The quantitative analysis of circular dichroic spectra of native human plasma fibronectin according to the method of Provencher and Glöckner [Provencher, S. W., & Glöckner, J. (1981) Biochemistry 20, 33-37] indicated the presence of beta-sheet (79%), beta-turn (21%), but no alpha-helix or random coil in the secondary structure. The calf alveolar heparan sulfates induced a change in the conformation of fibronectin: the magnitude of the change depended on the molecular properties of the particular heparan sulfate preparations.
Animals↗
Isolation of a novel cell-attachment and spreading-promoting protein from human serum.
A protein with potent cell-attachment and spreading-promoting activity was isolated from fibronectin-free human serum. The purification steps included affinity chromatography on heparin-agarose and preparative isoelectric focusing. The purified protein was homogeneous as judged from dodecyl sulphate/polyacrylamide-gel electrophoresis. It had an isoelectric point of 5.0 and an Mr of 52 000. The protein promoted the spreading of Chinese-hamster ovary cells to plastic in a manner similar to that observed with fibronectin.
Animals↗
Attachment of staphylococci and streptococci on fibronectin, fibronectin fragments, and fibrinogen bound to a solid phase.
The attachment of Staphylococcus aureus (Cowan I) and two strains of group A and G streptococci on glass cover slips coated with fibronectin, fibronectin fragments, or fibrinogen was studied. The attachment was quantitated by counting the attached bacteria on glass surfaces coated with a similar molarity of the proteins. Fibronectin was a more effective attachment factor than fibrinogen for staphylococci, while group G streptococci attached better on fibrinogen- than on fibronectin-coated cover slips. In this system, group A streptococci bound almost exclusively to substrate-bound fibrinogen. Attachment experiments involving the use of staphylococci pretreated with soluble fibronectin or fibrinogen revealed that bacterium-bound fibronectin and fibrinogen were able to enhance the adherence on cover slips coated with fibronectin. The 30-kilodalton NH2-terminal and the 120- to 140-kilodalton COOH-terminal fragments of fibronectin, both of which contain bacterial binding sites, mediated the staphylococcal attachment, suggesting that both parts of the molecule are involved in the attachment mediated by fibronectin.
Adhesiveness↗
Binding sites for streptococci and staphylococci in fibronectin.
Purified cathepsin G fragments of fibronectin were used to locate the binding sites for streptococci and staphylococci in the fibronectin molecule. The iodinated, NH2-terminal, 30-kilodalton (kd) fragment bound to group A and G streptococci and to Staphylococcus aureus. The 125I-labeled, COOH-terminal, 120- to 140-kd fragment bound weakly to group A streptococcus strain and to S. aureus when tested in a buffer of low ionic strength. The 30- and 120- to 140-kd fragments inhibited the binding of iodinated fragments to bacteria. The two fragments were, on a molar basis, equally effective, and they were more potent inhibitors than intact fibronectin. The gelatin-binding 40-kd fragment neither bound to any of the bacterial strains nor inhibited the binding of 125I-labeled 30-kd or 125I-labeled 120- to 140-kd fragments to bacteria. The results indicate that fibronectin has at least two separate binding sites for streptococci and staphylococci, one in the NH2-terminal region and another in the COOH-terminal region of the molecule, both capable of specific interaction with a complementary structure exposed on streptococcal and staphylococcal cell surfaces.
Binding Sites↗
Characterization of fibronectin on human spermatozoa.
Ejaculated human spermatozoa were shown to have fibronectin polypeptides on their surface. Immunofluorescence studies revealed fibronectin as a belt-like fluorescent band on the post-acrosomal area of sperm heads, whereas none was found in sperm tails. The location of the fluorescent band corresponded to the equatorial segment of the spermatozoon. Fibronectin polypeptides were heterogeneous with Mr ranging from 35000 to 210000, as revealed by immunoblotting and by immunoprecipitation of detergent extracts from surface-radioiodinated spermatozoa.
Electrophoresis, Polyacrylamide Gel↗
Identification of fibronectin fragments that bind to carboxy-group-modified proteins.
Limited proteolysis of human plasma fibronectin with chymotrypsin, trypsin or thermolysin has been used to localize binding sites responsible for binding [Vuento, Korkolainen & Stenman (1982) Biochem. J. 205, 303-311] of fibronectin to carboxy-group-modified proteins. These bindings sites are different from those mediating binding of fibronectin to gelatin or heparin. They are located close to the C-terminus of the polypeptide chains of fibronectin, and apparently overlap with the C-terminal fibrin binding site.
Binding Sites↗
Effect of chemical modification of arginine and lysine residues of fibronectin on its antigenic and gelatin-binding activity.
The effect of chemical modification of arginine and lysine residues of fibronectin on its antigenic and gelatin-binding activity was studied by enzyme immunoassay techniques. Both modifications strongly reduced the gelatin-binding activity. Using conformation-specific antibodies it was shown that modification of lysines caused extensive conformational changes in the molecule. No such changes could be detected in arginine-modified fibronectin. The results suggest that arginine residues are directly involved in the binding of fibronectin to gelatin. Lysine residues seem to be important for maintaining a native conformation necessary for gelatin-binding.
Antigens↗
Association of fibronectin with carboxy-group-modified proteins in vitro.
Treatment of human immunoglobulin G, albumin and fibronectin with water-soluble carbodi-imide at pH4.75 in the presence of glycine ethyl ester resulted in an avid binding of (125)I-labelled native fibrinectin to the modified proteins. Succinoylation, reduction and alkylation or heat-denaturation had no such effect. In affinity chromatography under physiological conditions, serum was depleted of fibronectin when run through columns of the carbodi-imide-treated proteins coupled to agarose. Fractions eluted from such columns with urea were enriched in fibronectin. The binding of radiolabelled fibronectin to the carbodi-imide-treated proteins was inhibited by unlabelled fibronectin in relatively low concentrations, but also by albumin in higher concentrations. Heat-denatured albumin inhibited at concentrations approx. 10-30 times lower than native albumin. The binding reaction had a pH optimum of 6-8. It was inhibited at high ionic strength and in the presence of urea. Anionic detergents inhibited at millimolar concentrations, but non-ionic detergents did not inhibit the binding reaction. The results were interpreted as showing that: (1) fibronectin is capable of binding to itself, to immunoglobulin G and to albumin after a reduction of the negative surface charge of these proteins, and may have a general ability to bind such modified proteins; (2) this binding can take place under physiological conditions; (3) carboxy-group-modified proteins selectively bind fibronectin from serum. This novel binding phenomenon could be important in terms of the opsonin function of circulatory fibronectin. We propose that fibronectin may recognize modified (denatured) proteins and mediate their uptake by the reticuloendothelial system.
Cations, Divalent↗
Essential charged amino acids in the binding of fibronectin to gelatin.
The binding of fibronectin to gelatin-agarose was strictly dependent on pH, having a pH optimum of 7-9. The binding was strongly inhibited by increasing ionic strength. A chemical modification of lysyl and arginyl groups of fibronectin abolished the binding activity. The anionic detergents sodium dodecyl sulphate and sodium deoxycholate in concentrations of 10-100mM had the same effect. The binding was not affected by the non-ionic detergents Triton X-100, Tween 20 or Lubrol WX. The results demonstrate an important role of ionic interactions in the binding of fibronectin to gelatin. Absence of inhibition by non-ionic detergents suggests that hydrophobic interactions contribute relatively little to the binding of fibronectin to gelatin.
Amino Acids↗
Fibronectin binds to charge-modified proteins.
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Blood Proteins↗
Competitive enzyme immunoassay for human plasma fibronectin.
A competitive enzyme immunoassay for the determination of fibronectin in plasma is described. An enzyme conjugate prepared by coupling alkaline phosphatase to rabbit anti-human fibronectin antibodies by glutaraldehyde was used as principal reagent. The assay was performed by coating polystyrene tubes with purified fibronectin and reacting these coated tubes with a mixture of sample and enzyme-labeled antibodies. After overnight incubation, the amount of enzyme activity associated with the tube was determined. An assay range of 0.5-20 microgram/ml of fibronectin was obtained. The mean concentration of plasma fibronectin in female patients was found to be 270 microgram/ml (standard deviation 50 microgram/ml, n = 22). Denatured fibronectin had low activity in the assay. The presence of cross-reacting antigens in rat and guinea pig plasma was demonstrated by the enzyme immunoassay technique.
Adsorption↗
Immunochemical characterization of human plasma fibronectin.
Human plasma fibronectin has been purified by a non-denaturing affinity chromatography procedure [Vuento & Vaheri, (1979) Biochem.J. 183, 331--337], and antisera have been raised by immunizing rabbits with the native protein. The antisera reacted strongly with native fibronectin, but only weakly with reduced and alkylated fibronectin or with heat-denaturated fibronectin. Denaturation also affected the haemagglutinating and gelatin-binding activities of fibronectin and increased its susceptibility to proteolytic degradation. The antisera reacted with fragments of fibronectin obtained by proteolysis with plasmin. Large fragments (mol.wt. 180000--200000), lacking the region harbouring the interchain disulphide bridges but containing the sites responsible for gelatin-binding and haemagglutinating activity, showed as intense a reaction with the antisera as intact fibronectin. Smaller peptides showed a weaker reaction. All fragments tested showed sensitivity to denaturation in their reaction with the antisera. The results were interpreted as showing that: (1) native fibronectin has an ordered conformation that is easily perturbed by denaturation; (2) most of the antigenic determinants of the protein are dependent on conformation; (3) the region of the fibronectin molecule containing the interchain disulphide bridges has only few antigenic determinants; and (4) covalent interaction of the two subunits does not contribute to the antigenic structure recognized by rabbit antisera. The observed correlation between the antigenic activity and a structural and functional intactness of fibronectin suggests that the antibodies to native fibronectin could be used as a conformational probe in studies on this protein.
Antigen-Antibody Reactions↗
Interaction of polyamines with proteins of human plasma: a preferential aggregation of fibrinogen and fibronectin (cold insoluble globulin).
The polyamines spermine and spermidine were found to aggregate proteins from human plasma and serum, apparently due to electrostatic interactions between these cations and anionic proteins. Fibrinogen and fibronectin (cold insoluble globulin) were identified as the major molecular components of aggregates, and fibronectin could be quantitatively removed from plasma by aggregation with spermine. The results indicate that fibrinogen and fibronectin have anionic groups which react with polyamines and which are essential for the solubility of these proteins.
Blood Proteins↗
Spontaneous and polyamine-induced formation of filamentous polymers from soluble fibronectin.
Fibronectin is a high-molecular-weight glycoprotein present in a soluble form in plasma and in other body fluids and as insoluble protein in connective tissue matrix. This study reports that soluble fibronectin is polymerized into filamentous structures and that polyamines stimulate this process and precipitate fibronectin. Fibronectin purified from human plasma under non-denaturing conditions appeared after negative staining as non-globular extended structures in the electron microscope. During storage of purified fibronectin at +4 degrees C, in particular a low ionic strength, increasing amounts of the protein appeared as protein filaments. These filaments had a diameter of 2--3 nm and a length of up to several micrometers. The filaments also formed bundles of variable thickness, apparently through lateral association. These structures could also be visualized by phase-contrast microscopy. Polyamines, at a concentration of 1--5 mM and at a low ionic strength, induced a rapid, extensive polymerization of fibronectin into filamentous structures. The effect increased in the order putrescine less than spermidine less than spermine. Polyamine-induced precipitation of fibronectin was reversible upon removal of the polyamine. Fibronectin secreted by normal and by malignant cells could be fairly selectively precipitated from the culture medium with polyamines. The observed filamentous polymers of soluble fibronectin resemble the filamentous fibronectin-containing pericellular structures in fibroblast cultures and may provide a model for studies on the deposition of fibronectin in matrix form.
Chromatography, Agarose↗
High concentrations of fibronectin-like antigens in human seminal plasma.
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Alkaline Phosphatase↗