Similarity of the carbohydrate moieties of fibronectins derived from blood plasma and synthesised by cultured endothelial cells

Plasma and cellular fibronectins are reported to be very similar but not identical in chemical structure. We have compared bovine plasma fibronectin with fibronectin secreted by bovine aortal endothelial cells in culture. Techniques were chosen to highlight likely structural differences, particularly in the carbohydrate moieties. Both fibronectins were wholly reactive to monospecific antiserum and behaved identically in two-dimensional gel electrophoresis. The oligosaccharide chains were identical in proportion and degree of sialylation by anion-exchange HPLC. Fractionation of the glycopeptides on immobilised lectins and serotonin showed that both fibronectins contained (i)_predominantly biantennary oligosaccharides, (ii) exclusively N-acetylneuraminic acid residues in a non-clustered array and (iii) no L-fucose residues. The overriding structural similarities support the proposal that the endothelium is a site of synthesis of plasma fibronectin in vivo.     

The carbohydrate components of corn prolamins]

This work was aimed to study the patterns of zein glycosylation. Zein proteins included 1-3% of sugars. The affinity of different lectins, such as concanavalin A (Con A), Lens culinaris lectin (LCL) and lectins of Arachis hypogaea (PNA), of Triticum vulgaris (WGA), of Dolichos biflorus (DBA), of Glycin max (SBA), of Lotus tetragonolobus (LTA), of Laburnum anagiroides (LAL), of Ricinus communis (RCA), of Phaseolus vulgaris (PHA) was used to analyze the glycosylation sites. All selected lectins interacted with zein proteins. It may serve a basis for determination of mannose, galactose, fucose and aminosugars. Some lectins were bound only by prolamines of some inbred lines, while others were connected with all lines.     

Methodology to label mixed carbohydrate components by APTS

Labelling of carbohydrates with fluorescent dyes is of importance in the analysis of complex oligo- or polysaccharides. The complexity of the hydrolization mixtures of the oligosaccharides can be studied with separation methods after labelling. However, the procedures might provide unexpected phenomena. The experimental circumstances for labelling carbohydrates with 8-aminopyrene-1,3,6-trisulfonic acid were studied and the labelling efficiency of mono and oligosaccharides present in endotoxins was followed by capillary electrophoresis using LIF detection. Significant differences were observed in the labelling of the different sugar molecules, and the lowest reactivity with the dye was observed in the cases of amino-sugars. The results provide a basis for studies in determining the structures of the core parts and the O-chains of bacterial endotoxins.     

Polypeptide heterogeneity of hamster and calf fibronectins.

The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide 'maps'. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344].     

Comparisons of evolutionarily distinct fibronectins: evidence for the origin of plasma and fibroblast cellular fibronectins from a single gene

Plasma and fibroblast cellular fibronectins from three different species were compared for structural similarities and differences. Partial tryptic digestion of either human or chicken plasma and cellular fibronectins yields homologous protease-resistant domains within a species but few homologies between species regardless of the source. Within a species, human or chicken plasma and fibroblast cellular fibronectins are immunologically indistinguishable as determined by the ELISA technique. There is limited immunological cross-reactivity between species. Two-dimensional tryptic peptide maps of fibroblast cellular and plasma fibronectins from the same species are also very similar: 85-95% of the spots on such maps comigrate. When peptide maps from different species are compared, no more than 10% of the spots comigrate. Three models for the genetic origin of cellular and plasma fibronectins in vertebrates are considered. A model in which both fibroblast cellular and plasma fibronectins arise from the same gene is the simplest that is consistent with the data.     

Immunolocalization of tenascin and cellular fibronectins in diverse glomerulopathies

Frozen samples of minimal change glomerulopathy (MCG), and of membranous, segmental and diffuse lupus glomerulonephritis (MGN, SGN, DLGN) were studied to assess the distribution of tenascin (Ten), and the extradomains A and B (EDA-and EDB-) and oncofetal (Onc-) isoforms of cellular fibronectin (cFn). Cryosections were immunostained by the ABC method with specific monoclonal antibodies. In MCG, mesangial Ten and EDA-cFn reactions were increased. In MGN, mesangial Ten and EDA-cFn staining was enhanced except in segmental scars; convincing reactions were seen in cases with membranous transformation; spikes stained strongly. In SGN, variably intense staining for Ten and all cFn isoforms was seen in glomerular necrosis, proliferation and crescents; parietal epithelium EDA-cFn staining was noted. In DLGN, strong and extensive mesangial Ten and EDA-cFn staining was seen as were focal EDB-and Onc-cFn reactions. Parietal cells with and without crescents stained variably with all Mabs. Obsolete glomeruli were unreactive save for rare periglomerular Ten rims. Interstitial inflammation and fibrosis in MGN, SGN and DLGN had moderate to strong Ten and EDA-cFn staining with rare traces of EDB-and Onc-cFn. We conclude that enhanced Ten and EDA-cFn is a potentially reversible response to glomerular injury whereas the expression of EDB-and Onc-cFn apparently result from necrosis and/or cellular proliferation which lead to scarring. And, while mesangial cells are the major source of these molecules, epithelial cells might also partake in their synthesis.     

The dimensionality of human's electroencephalogram during sleep

In order to perform an analysis of nonlinear EEG-dynamics we investigated the EEG of ten male probands during sleep. According to Rechtschaffen and Kales (1968) we scored the sleep-EEG and applied an algorithm, proposed by Grassberger and Proccaccia (1983) to compute the correlation dimension of different sleep stages. The correlation dimension characterizes the dynamics of the EEG signal and estimates the degrees of freedom of the signal under study. We could demonstrate, that the EEG of slow wave sleep stages depicts a dimensionality, which is two units smaller than that of light or REM sleep.     

Structural comparison of fibronectins from normal and transformed cells

Comparative study of the structures of fibronectins from normal and transformed cells by partial proteolysis as well as by tryptic peptide fingerprinting and analysis of phosphorylation show that: 1) fibronectin molecules from normal and transformed cells probably have very similar primary structures; 2) the phosphorylation of fibronectin is a highly specific and conserved phenomenon; 3) fibronectin from both normal and transformed cells is phosphorylated only on serine residues; 4) although the major sites of phosphorylation in fibronectin are the same in normal and transformed cells, fibronectin from transformed cells appears to be phosphorylated to a much higher extent than that from normal cells.     

Localization of carbohydrate components in rat colon with fluoresceinated lectins

Cryostat sections of rat descending colon were studied by fluorescence microscopy after exposure to conjugates of fluorescein isothicoyanate with lectins from Glycine max (soybean), Triticum vulgaris (wheat germ), Ricinus communis (castor bean), Ulex europaeus, (gorse), Dolichos biflorus (horse gram) and Canavalia ensiformis (concanavalin A) (Jack bean). No two lectins showed identical patterns of fluorescence. FITC-conjugates of soybean and D. biflorus lectins reacted strongly with the mucus present in the crypt lumens and with the surface (as well as cytoplasm) of the epithelial cells suggesting that these sites are rich in terminal, non-reducing, N-acetylgalactosamine residues. Wheat germ, R. communis, U. europaeus and concanavalin A-FITC conjugates did not stain mucus but showed fluorescence in the cytoplasm of absorptive cells as well as in the lamina propria and submucosa. The FITC-R. communis conjugate also reacted with structures in the apical portion of epithelial cells that may correspond to the Golgi apparatus.     

Structural and functional comparisons of chicken and human cellular fibronectins

We have investigated the structural and functional differences between chicken and human cellular fibronectin by comparing the tryptic peptide patterns using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analyzing the binding properties of isolated trypsin-resistant polypeptide fragments. Although the overall functional organization of chicken and human cellular fibronectins was similar, the tryptic patterns obtained from these two molecules were strikingly different. For example, the tryptic digest of chicken cellular fibronectin contained two unique peptide fragments having molecular sizes of 45 and 70 kilodaltons. The previously unidentified carboxyl-terminal 45-kDa fragment is an intermediate that appears between 15 to 120 s of digestion. The 70-kDa fragment binds to gelatin, to fibrin (with unusually high apparent affinity), to heparin (at low ionic strength), and to fixed Staphylococcus aureus cells; it also contains an acceptor site for factor XIIIa (plasma transglutaminase). These results suggest that the functional domains of chicken and human fibronectins remain constant and that structural variations occur in the protease-susceptible regions of the molecule. The present findings are discussed in terms of the previously existing discrepancies in the literature on fibronectin.     

Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens

Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12-28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30-66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.     

Purification of the influenza hemagglutinin glycoprotein and characterization of its carbohydrate components.

Hemagglutinin from influenza A/PR8 virus was purified after treatment of the virus with sodium deoxycholate followed by extraction with tri-n-butyl phosphate. This fully disrupted the virus while preserving hemagglutinating activity. The hemagglutinin was obtained in the form of small aggregates that could be separated from other viral components. Purified hemagglutinin was hydrolyzed to determine carbohydrate composition and digested with Pronase to analyze oligosaccharide structures. Sugars present in the hemagglutinin were galactose, mannose, fucose, and glucosamine in molar rates of about 6:11:2:5, and these comprised 16% of the hemagglutinin glycoprotein. Oligosaccharides obtained from virus included a major component of a molecular weight of 2,800, composed of glucosamine, galactose, mannose, and fucose, and a minor heterogenous component of a molecular weight of 1,500 to 2,000, containing predominantly mannose. The 2,800-molecular-weight oligosaccharide was a constituent of the hemagglutinin, and treatment of this large oligosaccharide with specific exo-glycosidases demonstrated the presence of terminal galactose and fucose and allowed the deduction of a general structure for this component.     

Seasonal dynamics of carbohydrate and nitrogenous components in the roots of perennial weeds

Abstract Chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale L.) are persistent weeds, the aerial portions of which do not survive in winter. However, subterranean tissues remain viable and facilitate the rapid resumption of growth in early spring. The source of nutrients for growth prior to the establishment of foliage is the roots. Carbohydrate and N reserves are accrued during late summer and autumn, respectively. Hydrolysis of fructans during late autumn occurs coincidentally with increments in sucrose, the latter providing a readily accessible C pool. Nitrate, free amino acids and soluble protein all play substantial roles in nitrogen storage. Asparagine is the predominant amino acid in the free pool during winter, followed by glutamine, ornithine, serine, aspartic acid and glutamic acid. Storage reserves remain at peak levels throughout winter and deeline prior to the resumption of growth. The patterns observed here provide evidence that N is an important currency of storage metabolism and, thus, a framework has been provided for the examination of regulation of N storage in perennial weeds.