Tapio Vartio

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.

Human gelatinase/type IV procollagenase is a regular plasma component

Gelatin zymograms revealed in human plasma a constant 66 kDa proteolytically active polypeptide. In most plasma samples other major proteolytic activities were seen at M r92 000,130 000 and 225 000. All four proteases were Ca2+‐dependent metalloproteases and bound quantitatively to gelatin‐Sepharose. Immunoblotting results indicated that the 66 kDa protease was the human fibroblast gelatinase/type IV procollagenase and that the other three proteases were macrophage/granulocyte‐derived gelatinase components. The 66 kDa protease did not bind to conA‐ nor lentil lectin‐Sepharose allowing its separation from the 92, 130 and 225 kDa proteases. During the isolation procedure the plasma gelatinase/type IV procollagenase tended to form a proteolytically active spontaneous disulfide‐bonded dimer and a 62 kDa component that could also be obtained by digestion with trypsin. The same polypeptide changes occurred also in stored preparations of the corresponding protease isolated from fibroblast culture medium while the freshly purified protein contained only the 66 kDa proform.

Fibronectin in synovial fluid and tissue in rheumatoid arthritis

Abstract. Fibronectin is a glycoprotein found in body fluids, loose connective tissue matrix and in basement membranes. Fibronectin in rheumatoid arthritis synovial fluid was immunologically indistinguishable from the plasma form, as shown by double‐diffusion analysis. Fibronectin isolated from rheumatoid synovial fluid by affinity chromatography on gelatin‐Sepharose had a polypeptide pattern similar to that of plasma fibronectin in SDS‐polyacrylamide gel electrophor‐esis. In fifty‐one patients with rheumatoid arthritis and related diseases fibronectin concentrations in synovial fluid were 445 ±103 μg/ml (mean ±SD) and within normal range, 335±52 μg/ml, in plasma. Immuno‐fluorescence staining showed a prominent increase of fibronectin in the proliferating synovial connective tissue in rheumatoid arthritis as compared to normal synovial membrane. The results suggest an increased local production of fibronectin in rheumatoid synovial tissue.

Cultured human amniotic fluid cells characterized with antibodies against intermediate filaments in indirect immunofluorescence microscopy.

Cells cultured from second trimester human amniotic fluid were characterized in indirect immunofluorescence (IIF) microscopy using specific antibodies against the subunit proteins of different types of cytoskeletal intermediate filaments. Most of the amniotic fluid cell cultures contained only epithelial cells as indicated by the positive keratin-fluorescence in IIF. Five distinct types of keratin-positive cells could be characterized. A dominating cell type (E-1) in most cultures were rapidly proliferating epithelial cells, previously called amniotic fluid cells (AF-cells). These cells showed a fibrillar cytoplasmic fluorescence both with keratin antibodies and with antibodies against vimentin, the fibroblast type of intermediate filament protein. E-1 cells did not show the typical cell-to-cell arrangement of keratin fibrils between the adjacent cells, a characteristic previously found in most cultured epithelial cells. Most of the cultures also contained large epitheloid cells (E-2), showing a fine fibrillar cytoplasmic organization of both keratin- and vimentin filaments, clearly different from that seen in E-1 cells. Several cultures contained two additional epithelial cells both showing the typical cell-to-cell arrangement of keratin fibrils (E-3 and E-4). These two cell types could be distinguished because of their distinct difference in size. E-4 cells typically grew as small cell islands among other epitheloid cells. Amniotic fluid cell cultures occasionally contained also large multinucleated cells (E-5), which appeared to contain large amount of fibrillar keratin. Fibroblastic cells, identified by their decoration only with antibodies against vimentin, were rarely found in amniotic fluid cell cultures. Interestingly, in such cultures some cells with a fibroblastoid appearance were identified as epithelial cells on the basis of the positive keratin-fluorescence. The results show the suitability of IIF with cytoskeletal antibodies in characterization of heterogenous cell populations and indicate that normal amniotic fluid cell cultures mostly contain epithelial cells.

Fibronectin: chains of domains with diversified functions

Abstract Fibronectin is now recognized to be a large, multifunctional, adhesive glycoprotein with a wide distribution in vivo . Other distinctive features of fibronectin include its domain structure and its various molecular and biological interactions thought to be involved in cell anchorage, elaboration of the extracellular matrix, hemostasis, chemotaxis and opsonization. Structural studies of the protein are indicating that the function correspond to specific interactive domains in the molecule and are revealing unique, internally homologous sequences in the primary structure. Many of the biological effects of fibronectin are shared by its proteolytic fragments which, interestingly, also seem to have effects of their own.

A novel lectin‐independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin

Binding of P fimbriae of uropathogenic Escherichia coli to purified human plasma fibronectin and human placental type IV collagen was studied. In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30‐kDa fragment and the 120–140‐kDa carboxyterminal fragments of fibronectin. Binding to the gelatin‐binding 40‐kDa fragment of fibronectin was considerably weaker. No binding to immobilized type IV collagen was seen. The interaction between P fimbriae and immobilized fibronectin was not inhibited by α‐D‐Gal‐(1‐4)‐β‐D‐Gal‐1‐O‐Me, a receptor analog of P fimbriae. Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays. Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble 125I‐labelled fibronectin was found. The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the fimbriae.

Enrichment of A 140 KD surface glycoprotein in adherent, detergent-resistant cytoskeletons of cultured human fibroblasts

Abstract Radioactive surface and metabolic labeling techniques were used to study the surface glycoproteins which remain in detergent-resistant cytoskeletons of cultured human fibroblasts. A 140 kilodalton (kd), fucose-containing glycoprotein (gp) was enriched in the cytoskeletal preparations together with extracellular matrix fibronectin. The 140 kd gp resisted trypsintreatment and was present as a major surface glycoprotein also in cytoskeletons of newly attached cells which had deposited only minimal quantities of extracellular matrix. In isoelectric focusing the protein was separated into three spots. The results suggest that the 140 kd gp is a cytoskeleton-associated surface glycoprotein which may play a role in the attachment of the cytoskeleton to the growth substratum.

Monoclonal antibodies in analysis of cathepsin G-digested proteolytic fragments of human plasma fibronectin

Proteolytic fragments of fibronectin, obtained by digestion with cathepsin G, were transferred electrophoretically from sodium dodecyl sulphate (NaDodSO4) polyacrylamide gels to nitrocellulose sheets and used as antigens for monoclonal antibodies. All 9 monoclonal antibodies tested reacted with undenatured intact fibronectin or its fragments applied directly to nitrocellulose sheets. Two of the clones did not react with the NaDodSO4-treated transferred material suggesting reactivity with conformational determinants. Distinct fragments of fibronectin could be detected by several of the antibodies. None of the monoclonal or the polyclonal antibodies used reacted with the Mr = 40,000 or Mr = 30,000 gelatin-binding fragments of fibronectin. However, one of the monoclonal antibodies reacted specifically with their precursor Mr = 64,000 fragment, but apparently with its gelatin-nonbinding segment. The apparent non-immunogenicity of the gelatin-binding domain is conspicuous, suggesting that it may be highly conserved in evolution. The present method, combination of controlled proteolytic cleavage with electrophoretic transfer, provides an effective means for characterization of monoclonal antibodies raised against proteins.

Polypeptides of human plasma fibronectin are similar but not identical

Human plasma fibronectin migrated in electrophoresis after reduction of the disulfide bonds in SDS-polyacrylamide gels as two closely spaced polypeptide bands. These polypeptides, the A chain (Mr 220 000) and the B chain (215 000), were isolated from slices of slab gels. The two isolated chains were immumologically indistinguishable when tested by double immunodiffusion against antiserum to plasma fibronectin. Identical peptides were obtained from both chains after Staphylococcus aureus proteinase digesting or after cyanogen bromide cleavage, respectively. Kinetic analysis of plasmin digestion of isolated dimeric fibronectin, however, suggested that the A chain was cleaved more rapidly than the B chain and that the primary plasmin cleavage products of fibronectin, the 200 000 and 190 000 polypeptides, were derived from the A and B chain, respectively. The basis for the difference between the A and B chains of human plasma fibronectin identified here, is, at present, unknown. Proteolytic or some other posttranslational processing of a common fibronectin polypeptide seems unlikely since also the newly synthesized fibronectin, isolated from human fibroblast cultures pulse-labeled for 5 min, appeared as two closely spaced polypeptide bands in SDS-gel electrophoresis.

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.