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Biomedical subjects

T Vartio

Publications and source records attributed to T Vartio.

At least 55 records · Page 3Linked to original sources
PubMed1985

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↗
PubMed1985

The Mr 95,000 gelatin-binding protein in differentiated human macrophages and granulocytes.

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well-characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross-reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin-binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.

Adult↗
PubMed1984

A rapid and highly sensitive solid-phase enzyme immunoassay specific for human fibronectin using a characterized monoclonal antibody.

A solid-phase enzyme immunoassay (EIA) was developed for the quantitation of human fibronectin in body fluids and cell culture media. In the assay a human fibronectin-specific murine monoclonal IgG1 (f-33) was used as capture antibody and polyclonal rabbit anti-fibronectin as detector antibody. The antibody showed no reactivity to purified monkey, dog, rabbit, horse, sheep, mouse, bovine or chicken fibronectins. The determinant of the monoclonal antibody was mapped to the cell-binding region of the fibronectin molecule. This localization was based on the use of purified fragments of fibronectin, immunoblotting, EIA and inhibition of fibroblast adhesion and spreading by the antibody. The detection limit of the fibronectin assay was 2 ng/ml. The assay was used for the quantitation of fibronectin in human plasma, urine and cerebrospinal fluid specimens and culture media of human cells.

Animals↗
PubMed1984

Tissue type plasminogen activator, but not urokinase, exerts transformation-enhancing activity.

Previous studies have established that plasma cryoprecipitates of tumor patients, culture media of transformed cells and defined proteolytic fragments of fibronectin enhance the morphological cell transformation ( TEF activity) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We now report that purified human tissue type plasminogen activator (t-PA), but not urokinase (u-PA), has a similar TEF activity, at doses as low as 2 ng/ml (30 pM). Specific antibodies effectively neutralized the activity. No significant contamination (less than or equal to 1%) between the preparations of t-PA and fibronectin (FN) or its fragments ( FNdp ) was detected. The results suggest that t-PA may have a direct role in the process of morphological cell transformation in vitro.

Cell Transformation, Neoplastic↗
PubMed1984

Localization of viral-envelope-glycoprotein-binding sites in fibronectin.

Purified viral-envelope glycoproteins from influenza A virus were found to bind to two fragments of the fibronectin molecule. Human plasma fibronectin was digested by leucocyte cathepsin G, and three different fragments, of Mr 30000, 40000 and 12000-140000, with specific binding functions were isolated. Micelles of radiolabelled influenza A glycoprotein were allowed to bind to these fragments immobilized on polystyrene micro-titre wells. The C-terminal 120000-14000-Mr fragments that carry the cell-binding activity bound viral proteins most efficiently, whereas the 40000-Mr gelatin-binding fragment bound considerably less. The N-terminal 30000-Mr Staphylococcus aureus-binding fragment was negative in the assays. Laminin, a basement-membrane protein, also bound viral proteins, though less effectively than fibronectin. The binding was abolished if laminin or fibronectin fragments were pretreated with neuraminidase. This suggests that the sialic acids in the sugar moieties of these glycoproteins are involved in the binding. The affinity of viral-envelope glycoproteins for certain domains of fibronectin and for laminin may play a role in virus-cell interactions.

Binding Sites↗
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↗
PubMed1984

Integrity of the pericellular fibronectin matrix of fibroblasts is independent of sulfated glycosaminoglycans.

The pericellular matrix fibers of cultured human fibroblasts contain fibronectin, other glycoproteins, and heparan and chondroitin sulfate proteoglycans. In the present study, cell-free pericellular matrices were isolated from metabolically labeled fibroblast cultures. The isolated matrices were digested with heparinase from Flavobacterium heparinum, and then analyzed for sulfated glycosaminoglycans (GAGs). Nitrous acid degradation was used to distinguish the N-sulfated GAGs (heparan sulfate) from chondroitin sulfate. Fibronectin and the other major matrix polypeptides were studied using gel electrophoresis, enzyme immunoassay and immunofluorescence. Upon heparinase digestion, greater than 95% of sulfated GAGs were degraded in the matrix without detectable release of fibronectin or other matrix polypeptides or alteration of the fibrillar matrix structure. We conclude that in fibroblast cultures the integrity of the fibrillar matrix is independent of sulfated GAGs. Together with earlier observations, this suggests that filamentous polymerization of fibronectin forms the backbone of early connective tissue matrix.

Cells, Cultured↗
PubMed1984

Cellular differentiation of basal cell carcinoma studied with fluorescent lectins and cytokeratin antibodies.

The expression of cellular glycoconjugates and cytokeratin polypeptides in 8 basal cell carcinomas (BCC) was studied using fluorochrome-coupled lectins and different keratin-antibodies. Peanut agglutinin and Wistaria floribunda agglutinin, binding to all layers of normal human epidermis, also stained all cells in the basal cell carcinomas. Dolichos biflorus agglutinin, which in normal epidermis binds only to the basal cells, gave a mottled staining pattern in most of the tumors. Instead, Ulex europaeus I agglutinin and soybean agglutinin, which in normal epidermis only bind to the spinous and granular cell layers, did not stain tumor cells in basal cell carcinomas. Rabbit antibodies to human 43-50kD epidermal keratin polypeptides and 2 monoclonal cytokeratin antibodies, PKK1 reacting only with follicular epithelium, and PKK2 reacting also with the basal epidermal cells, brightly stained all cells of the basal cell carcinomas studied, whereas antibodies to human 60-67kD epidermal keratin polypeptides did not bind to the carcinoma cells. The results suggest that the cells in basal cell carcinomas resemble epidermal basal cells both by their glycoconjugate pattern and keratin expression. However, the tumor cells also express cytokeratins, which can be found only in the follicular epithelium, but not in normal interfollicular epidermis.

Adult↗
PubMed1984

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↗
PubMed1984

Low doses of brotizolam and nitrazepam as hypnotics in the elderly.

0.125 mg brotizolam and 2.5 mg nitrazepam proved to be equipotent in promoting and maintaining sleep in 29 elderly patients used to long-term hypnotics. These drugs were superior to placebo and they were excellently tolerated. The drugs were administered in a random order, double-blind, for 7 nights each.

Aged↗
PubMed1983

Monoclonal antibody against the N-terminal end of human plasma fibronectin.

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.

Antibodies, Monoclonal↗
PubMed1983

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them.

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.

Antibodies, Monoclonal↗
PubMed1983

Expression of cytokeratin polypeptides in mouse oocytes and preimplantation embryos.

The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos.

Animals↗
PubMed1983

Characterization of a detergent-resistant surface lamina in cultured human fibroblasts.

Treatment of cultured human fibroblasts with 0.5% Triton X-100 produces substratum-anchored cytoskeletal preparations consisting of cytoplasmic filaments, nucleus and a plasma membrane-derived surface lamina. The lamina was visualized in fluorescence microscopy with fluorochrome-coupled wheat germ agglutinin (WGA) as a lace-like structure, extending throughout the cell domain. It displayed a different organization at the ventral and dorsal surfaces of the cell, partially coaligning with bundles of actin and myosin filaments at the dorsal cell surface. At the ventral surface vinculin patches appeared to be included in the surface lamina. Polyacrylamide gel electrophoresis, combined with lectin reactivity studies and lectin affinity chromatography, revealed a 140 kD sialoglycoprotein as the major glycoprotein component of the surface lamina.

Cell Membrane↗
PubMed1983

Microfilaments and intermediate filaments in epithelial cells transformed by murine sarcoma or leukemia viruses.

The murine epithelial cell line MMC-E was used to study changes in the cytoskeletal organization associated with viral transformation of epithelial cells by two different viruses. The cells were transformed with Moloney mouse sarcoma virus (MSV) or murine leukemia virus (MuLV). The expression of actin, myosin and of intermediate filament proteins in the cells was then studied. In MMC-E cells actin and myosin were organized as belt-like structures at the edges of the border cells of the cell islands and also circumferentially in the cells inside the islands. The major change after transformation was the decrease of the actomyosin containing belt extending from cell to cell at the borders of the cell islands. Both MMC-E cells and the MSV-transformed cells contained keratin as a juxtanuclear granular aggregate whereas the MuLV-transformed cells showed bright fibrillar arrays of keratin. Both virus-transformed cell lines showed enhanced vimentin-specific fluorescence and analysis of their cytoskeletal polypeptides confirmed the result. Similar molecular forms of keratin polypeptides were seen in all cells by immunoblotting. Viral transformation of MMC-E epithelial cells thus leads to different changes in their cytoskeletal organization depending on the transforming viral or cellular gene.

Actin Cytoskeleton↗
PubMed1983

Fibronectin: multiple interactions assigned to structural domains.

Characteristic features of fibronectin are its various molecular and biological interactions and a wide distribution in vivo. Structural studies of the protein have indicated that the functions correspond to specific interactive domains in the molecule and have revealed unique, internally homologous sequences in the primary structure. Fibronectin is easily degraded by proteinases and many of its biological effects are shared by the proteolytic fragments which also seem to have effects of their own.

Amino Acid Sequence↗
PubMed1983

Fibronectin and its proteolytic fragments. Potential as cancer markers.

Since the discovery of fibronectin as a transformation-sensitive 'cell surface' protein, it has been the focus of intensive studies. Fibronectin has multiple interactions and functions and is composed of distinguishing properties of malignant cells, properties. Invasiveness and metastasis, distinguishing properties of malignant cells, involve penetration through components of the extracellular matrix. Enzymatic degradation of matrix components is involved in these phenomena. Proteolytic targets of the pericellular matrices of cells in culture include fibronectin that has been found to be even selectively susceptible to proteinases. Defined fragments of fibronectin have transformation-promoting activity in experimental conditions and such fragments, detected in tumor patient body fluids, may serve as markers for tumor progression.

Amino Acids↗