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Biomedical subjects

T Vartio

Publications and source records attributed to T Vartio.

At least 19 recordsLinked to original sources
PubMed1992

Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator.

Regulation of the activity of proteolytic enzymes is of major importance in the turnover of connective tissues. The search for physiologically relevant activation mechanisms of principal tissue-degrading enzymes, e.g., metalloproteinases, has therefore been of wide interest. We have now studied whether the initiating factor of the fibrinolytic system, urokinase plasminogen activator (u-PA), may also function in the early steps of activation of one of the metalloproteinases, the M(r) 72,000 gelatinase/type IV collagenase produced by cultured fibroblasts. Treatment of the secreted M(r) 72,000 proteinase by u-PA yielded a cleavage product of M(r) 62,000 as revealed by fluorography of radioactively labeled proteins as well as by gelatin zymography SDS-PAGE gels. The u-PA-catalyzed cleavage of the M(r) 72,000 proteinase was blocked by anti-u-PA antibodies, but was unaffected by the plasmin inhibitor aprotinin, thus indicating a specific action for the activator. On the contrary, the tissue activator of plasminogen, t-PA, did not cleave the type IV collagenase in similar assays. u-PA-catalyzed cleavage of recombinant type IV collagenase, produced in a baculovirus expression system, yielded a similar M(r) 62,000 activity in gelatinolysis assay. Zymograms of the isolated pericellular matrices of cultured fibroblasts also revealed M(r) 72,000 gelatinolytic polypeptide that was converted to an M(r) 62,000 form by u-PA. Both polypeptides were recognized in immunoblotting by antibodies against the gelatinase/type IV collagenase, suggesting immunological identity with the secreted enzyme. Thus the M(r) 72,000 gelatinase/type IV collagenase is not only secreted, but also deposited into the pericellular fibroblast matrix, and both forms are substrates for u-PA. The results suggest a new potential role for u-PA as a direct regulator of metalloproteinase-mediated extracellular proteolysis via the cleavage of the M(r) 72,000 gelatinase/type IV collagenase to an M(r) 62,000 form.

Cell Line↗
PubMed1992

Adhesion protein YadA of Yersinia species mediates binding of bacteria to fibronectin.

The interaction between fibronectin and Yersinia strains was studied. Wild-type Y. enterocolitica strains expressing the virulence-plasmid-encoded adhesion protein YadA adhered strongly to fibronectin-coated coverslips, while their plasmid-cured variants expressed weaker binding. The cloned yadA gene of Y. enterocolitica or Y. pseudotuberculosis conferred fibronectin-binding ability both to Escherichia coli and to Y. psuedotuberculosis strains lacking the YadA protein. The YadA protein did not mediate binding to isolated fragments of fibronectin or to soluble fibronectin.

Adhesins, Bacterial↗
PubMed1991

Disulfide-bonded dimerization of fibronectin in vitro.

Human plasma fibronectin was denatured with 8 M urea and reduced with dithiothreitol. Dialysis or dilution of the solution led to formation of fibronectin dimers which migrated in non-reducing SDS/PAGE similarly to untreated control protein. When the redimerized fibronectin was reduced and re-electrophoresed it formed a doublet of alpha and beta chains of equal intensity indicating that it was a heterodimer. Low concentrations (less than 1 mM) of Fe3+ enhanced the redimerization of fibronectin, suggesting that metal ions may mediate oxidative reactions in the formation of the disulfides. Consequently, redimerization of fibronectin was completely prevented by deferoxamine, an iron chelator. Dimerization of fibronectin took place most effectively at pH greater than or equal to 8.8 but decreased strongly at lower pH, representing more unfavourable conditions for the action of the thiolate anion in the thiol/disulfide exchange reaction. Redimerized fibronectin, however, lost many of its binding properties to macromolecular ligands, suggesting that the disulfide bonding did not entirely regenerate the proper conformation of the protein. Pulse/chase experiments of fibroblast cultures showed that the initially monomeric fibronectin was rapidly and quantitatively dimerized under conditions representing natural pH and environment. SDS/PAGE analysis of the dialyzed urea-denatured/reduced thrombin and plasmin digests of fibronectin revealed that the NH2-terminal 30-kDa fragment and other fragments that contained intrachain disulfides quantitatively regained their non-reduced electrophoretic mobility. The results show that the dimerization and formation of intrachain disulfides of fibronectin may occur, in part, spontaneously, based on the amino acid sequence information of the protein. However, complete disulfide formation may also need other factors, present only in living cells, as suggested by pulse/chase experiments in fibroblasts.

Cells, Cultured↗
PubMed1991

Human natural killer cells express different integrins and spread on fibronectin.

Human natural killer (NK) cells adhered and most of them also actively spread on cellular fibronectin (cFn), plasma Fn (pFn) and its Mr 120,000-140,000 or Mr 105,000 cell-binding proteolytic Fn-fragments as well as on heparin-binding Fn-fragments containing the alternative cell binding site. The cells did not spread on vitronectin, laminin or collagens. Adhesion on Mr 105,000 Fn fragment containing the cell binding site, could be prevented by the synthetic peptide GRGDS but not by an inactive peptide, whereas adhesion on heparin-binding Fn fragments was unaffected by the peptide. Spreading of the NK cells led to a distinct reorganization of F-actin. Immunoprecipitation with monoclonal antibodies (MoAb) against the beta 1 integrin subunit of radioactively surface-labelled cells revealed a broad polypeptide band of Mr 140,000 under reducing conditions and a polypeptide doublet of Mr 160,000 and Mr 110,000 under non-reducing conditions. Identical polypeptides, corresponding to the alpha- and beta-subunits of the Fn-receptor complex, were bound to the Mr 105,000 chymotryptic Fn-fragment coupled to Sepharose. Similar experiments with small lymphocytes did not reveal any polypeptides. Immunofluorescence results with McAbs suggested that among the alpha-subunits of integrins, the alpha 3, alpha 4, and alpha 5 subunits are expressed in NK cells. The present results suggest that non-activated NK cells, but not small lymphocytes, express beta 1-integrins, and that at least the Fn-receptors alpha 4 beta 1 and alpha 5 beta 1 may function in the adhesion and migration of NK cells.

Cell Adhesion↗
PubMed1991

Cellular fibronectins are differentially expressed in human fetal and adult kidney.

The localization of cellular forms of fibronectin (cFn) was studied in fetal and adult kidneys. We used monoclonal antibodies reacting with the extradomains A and B in cFN (EDAcFn, EDBcFn) as well as with a differentially glycosylated fetal form of the protein (Onc-cFn). In adult human kidney EDAcFn was present in glomerular mesangium and in the walls of larger blood vessels, whereas a polyclonal rabbit fibronectin antiserum widely reacted also with interstitial areas. Immunoreactivity for EDBcFn and Onc-cFn, however, was not found in adult kidney. In the basement membranes and interstitial areas of developing tubules and glomeruli the immunoreactivity for EDAcFn was distinct and detectable in the earlier stages also for EDBcFn. In developing glomeruli, EDAcFn and EDBcFn were detected in teh mesangial areas, but in more mature fetal glomeruli, the mesangial immunoreactivity only persisted for EDAcFn. Both EDAcFn and EDBcFn were found in the basement membranes in the medullary area of all developing kidneys. In fetal kidney, immunoreactivity for EDAcFn and EDBcFn was seen also in small blood vessels, including the capillaries. Immunoreactivity for Onc-cFn was found in mesangial cells of fetal glomeruli as well as in the intima of larger blood vessels but not in the basement membranes. The results show that the three forms of cFn are present in the fetal kidney and have certain differences in their distribution. Conversely, only the EDAcFn was detected in the adult kidney. The different, partially age-related distributions of the three types of cFns suggest that they may also differ in their functions.

Adult↗
PubMed1990

Cellular fibronectin in rheumatoid synovium and synovial fluid: a possible factor contributing to lymphocytic infiltration.

Mouse monoclonal antibodies against ED sequence-containing cellular fibronectin (cFn) were used to show that Fn in the inflamed synovium is distinct from the major form of plasma Fn (pFn). An accumulation of cFn was seen at sites of hyperplasia of the rheumatoid synovial membrane and in the walls of small vessels in the synovium by immunofluorescence microscopy. cFn was also found in rheumatoid synovial fluid by immunoblotting. Approximately one-fifth of the T lymphocytes from rheumatoid synovial fluid bound to Fn. The binding of synovial fluid T cells was always higher than that from peripheral blood. These results have two implications. On the one hand, the cellular type of Fn may be an indicator of synovial inflammation. On the other hand, the deposition of Fn may be a factor contributing to the infiltration of mononuclear cells into the synovium.

Animals↗
PubMed1989

Interaction of Campylobacter jejuni with extracellular matrix components.

The adhesion of three strains of Campylobacter jejuni to coverslips and microwells coated with isolated extracellular matrix components, fibronectin, laminin and types I, III, IV and V collagens was studied. Fibronectin mediated the adherence of C. jejuni, but there were differences in the binding capacities of the strains. Type I, III and V collagens mediated very strongly the attachment of two strains of C. jejuni. All three strains attached weakly to basement membrane-specific type IV collagen. Laminin was capable of mediating the adhesion only when present at a higher concentration. The observations indicate that extracellular matrix components may serve as anchor molecules for C. jejuni adhesion and that several attachment mechanisms occur simultaneously.

Bacterial Adhesion↗
PubMed1989

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 Mr 92,000, 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.

Collagenases↗
PubMed1989

Regular fragmentation of hydrogen peroxide-treated fibronectin.

In the presence of low concentrations (less than 0.5 mM) of hydrogen peroxide Mr 350,000 and 170,000 fragments were generated from plasma and fibroblast medium fibronectins (Fns). No other major fragments were detected when H2O2 concentration was raised or the incubation time prolonged. A 200-300-fold concentration of H2O2 was needed for a complete degradation of the protein. The degradation was inhibited or completely prevented by deferoxamine, diethylene-triaminepentaacetic acid, and thiourea or by Chelex-pretreatment of the Fn solution suggesting a Fenton-type reaction to produce .OH radicals from H2O2. In immunoblotting the Mr 170,000 fragment reacted with monoclonal antibodies against the NH2 terminus and mid-molecule but not with those against the cell-binding site and the COOH terminus of Fn. Reduction of the Mr 350,000 fragment produced alpha- and beta-monomers of Fn as well as Mr 95,000 and 85,000 fragments which reacted with monoclonal antibodies against the cell-binding site and the COOH terminus of Fn. These results suggest that the Mr 170,000 fragment is derived from the NH2-terminal part of both subunits of Fn. The rest of the subunits, the Mr 95,000 (from alpha-chain) and Mr 85,000 (from beta-chain), thus remain disulfide-bonded to an intact Fn subunit to form the nonreduced Mr 350,000 polypeptide. The results show that oxygen radical action may generate defined and reproducible fragments from Fn. The high susceptibility of Fn to the radical induced degradation makes it plausible to occur also in vivo.

Binding Sites↗
PubMed1989

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 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 alpha-D-Gal-(1-4)-beta-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.

Bacterial Adhesion↗
PubMed1989

Embryonal carcinoma cells adhere preferentially to fibronectin and laminin but their endodermal differentiation leads to a reduced adherence to laminin.

F9 and PC13 embryonal carcinoma (EC) cells adhered rapidly to growth substrata coated with fibronectin or laminin. When F9 cells were induced to differentiate into visceral or parietal endoderm-like cells, their ability to adhere to laminin diminished, but their adherence to fibronectin remained unchanged. Correspondingly, permanently differentiated teratocarcinoma-derived endoderm cells (PYS-2 and PSA-5e) adhered markedly less efficiently to laminin than to fibronectin. F9 cells adhered to proteolytic fibronectin fragments containing the cell-binding site but not to fragments containing gelatin- or heparin-binding sites. They also adhered slowly to gelatin, but this adhesion was completely blocked by cycloheximide. The results show that the teratocarcinoma stem cells may have specific mechanisms mediating adhesion to fibronectin and laminin and that endodermal differentiation leads to a reduction in their capacity to adhere to laminin but not to fibronectin.

Cell Adhesion↗
PubMed1989

ED-A sequence containing fibronectin in human amniotic fluid and amnion epithelial cells.

1. Human amniotic fluid fibronectin (aFn) was studied by using a monoclonal antibody 52DHl (DH) that recognizes the extra domain (ED-A) sequence of cellular Fn (cFn). 2. In immunoblotting the DH antibody reacted with a sharp polypeptide band at the top of the bulk of the diffuse aFn. Another monoclonal antibody 52BF12 (BF) against the cell binding site of Fn, recognized the whole aFn. 3. The ED-A sequence containing cFn (EcFn) formed a constant proportion in aFns from all amniotic fluid preparations studied. 4. In amniotic membranes the DH antibody revealed bright subepithelial immunofluorescence. 5. Also isolated and cultured human amnion epithelial cells were strongly positive in immunofluorescence and secreted EcFn into the culture medium as revealed by immunoblotting. 6. The results indicate that aFn is a composition of at least two different Fn subtypes of which the EcFn most probably originates from amnion epithelial cells.

Amnion↗
PubMed1989

The O75X adhesin of uropathogenic Escherichia coli is a type IV collagen-binding protein.

Interaction of the basement-membrane binding O75X adhesin of uropathogenic Escherichia coli with various extracellular matrix proteins was studied. The adhesin showed strong binding to type IV collagen immobilized on microtitre plates, whereas other collagens, laminin and fibronectin, were only weakly recognized. Similarly, specific binding of [125I]-labelled type IV collagen to O75X-positive bacteria was shown. Interaction of the two proteins was also demonstrated by affinity chromatography of the O75X adhesin on immobilized type IV collagen. The adhesin bound strongly to the immobilized N-terminal 7S domain of type IV collagen, and the binding of [125I]-labelled type IV collagen to O75X-positive bacteria was inhibited by the soluble 7S domain. Binding of O75X to type IV collagen and to its 7S domain was specifically inhibited by chloramphenicol but was not affected by periodate or endoglycosidase-H treatment of the glycoproteins. Our results show that the 7S domain of type IV collagen is the basement membrane receptor for the O75X adhesin and suggest an interaction based on protein-protein recognition. Inhibition of the interaction by chloramphenicol favours the supposition that a modified tyrosine is involved in the binding site.

Acetylglucosaminidase↗
PubMed1989

Preparative polyacrylamide gel electrophoresis in the isolation of the nephritogenic proteins of passive Heymann nephritis.

To study kidney antigens involved in the formation of glomerular subepithelial immune deposits in passive Heymann nephritis polypeptides of 500, 130 and 105 kDa were isolated from rat kidney brush border (BB) membrane fraction using preparative polyacrylamide gel electrophoresis. Polyclonal antibodies raised against these proteins were specific for their respective antigens in immunoblotting. All three antisera bound to proximal tubular BB of kidney and to apical surfaces of several other epithelia as shown by indirect immunofluorescence on frozen sections of normal rat tissues. The anti-500 kDa and anti-105 kDa, but not the anti-130 kDa, antibodies also stained glomeruli and the anti-105 kDa antibodies also endothelial cells. After injection into rats the anti-500 kDa IgG bound to kidney glomeruli forming diffuse, granular deposits of rabbit IgG along the glomerular capillary walls, as shown by direct immunofluorescence. In electron microscopy the immune deposits were subepithelial and electron dense. The deposits remained in glomeruli for at least 60 days and increased with time. Deposits of C3 were not detected and proteinuria did not develop. The anti-130 kDa and the anti-105 kDa IgGs did not form glomerular deposits after in vivo injections. The results suggest that the 500 kDa and the 105 kDa proteins or related antigens are present in glomeruli and the 500 kDa protein is located on the epithelial side of the glomerular basement membrane. Circulating antibodies can bind to the 500 kDa protein forming immune complexes which rearrange and form electron dense deposits. The results further demonstrate that preparative gel electrophoresis is a useful technique for the isolation of kidney proteins of immunopathologic interest.

Animals↗
PubMed1989

An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis.

Fibroblasts in healthy adult lung are quiescent, synthesizing little collagen. We studied lung biopsies from 30 patients with pulmonary fibrosis, using immunohistochemistry with monoclonal antibodies against the propeptides of type I collagen to localize fibroblasts actively synthesizing collagen. Adjacent sections were stained with antibodies to type III and IV collagen, fibrin, cytokeratin, plasma fibronectin, or EDIIIa-containing "cellular" fibronectin (cFN). In rapid pulmonary fibrosis, including the proliferative phase of diffuse alveolar damage, organizing pneumonia, and subacute idiopathic fibrosis, collagen-synthesizing cells were numerous in organizing exudate filling airspaces but were also seen in the interstitium of the alveolar walls, interlobular septa, and walls of blood vessels. The new matrix deposited in the airspaces also contained type III collagen and EDIIIa-containing fibronectin. In chronic pulmonary fibrosis, more than half of the biopsies showed foci of collagen synthesis and cFN deposition near the air-tissue interface. The foci were consistently localized outside remnants of basal lamina and therefore within airspaces. The results indicate that (1) fibrosis in chronic idiopathic pulmonary fibrosis results mainly from organization of exudate within airspaces, just as it does after acute lung injury, and (2) during this process, fibroblasts increase their synthesis of collagen and fibronectin coordinately. Foci of active matrix deposition provide evidence for the progressive nature of chronic pulmonary fibrosis.

Antibodies, Monoclonal↗
PubMed1988

Differential expression of the extra domain-containing form of cellular fibronectin in human placentas at different stages of maturation.

The distribution of the extra domain-containing form of cellular fibronectin was studied in human placentas at different stages of maturation by using the monoclonal antibody 52DH1 in indirect immunofluorescence. In early chorionic tissue (7 to 10 weeks post menstruationem) cellular fibronectin was codistributed with laminin and type IV collagen in the trophoblastic basement membranes. At weeks 11 to 12 the trophoblastic basement membranes were negative but positivity was typically revealed in distinct aggregates in the stromal tissue. In second-trimester and term placentas the immunoreactivity was confined to the vessel endothelia of villous stroma. Extravillous trophoblast cells seen in placentas at different stages did not show positivity. Double staining with the 52DH1 monoclonal antibody and polyclonal fibronectin antibodies showed that both in the early and term placentas there was much fibrillar positivity only revealed with the polyclonal antibodies. The present results show that cellular fibronectin is a prominent component of early trophoblastic basement membranes and may thus play a special role in the maturation of chorionic villi.

Antibodies, Monoclonal↗
PubMed1988

Platelet glycoprotein IIb/IIIa complex in cultured cells. Localization in focal adhesion sites in spreading HEL cells.

A panel of mouse monoclonal antibodies (MoAbs) was raised that react with platelet glycoproteins (GP) IIb or IIIa. On immunofluorescence, the MoAbs reacted with 30% to 40% of the human erythroleukemia (HEL) cells. When the HEL cells were induced to spread on fibronectin in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), the MoAbs reacted with the focal adhesion sites. Only some of the GPIIIa MoAbs reacted with cells other than platelets, megakaryocytes, or HEL cells, and these showed focal adhesion sites in cultured human endothelial cells, fibroblasts, and epithelial cells from normal kidney tubules. They did not react, however, with transformed fibroblasts, fibrosarcoma cells, cultured cells from hypernephromas, or cultured human amnion epithelial cells. The results suggest that the platelet-type GPIIb/IIIa complex is only expressed in cells showing an ability to define megakaryoblastic differentiation. Localization of the GPIIb/IIIa complex at the induced focal adhesion sites in HEL cells and localization of the GPIIIa-like molecules in other cells suggest their direct role in the adhesion process and in the actomyocin organization of adherent cells.

Antibodies, Monoclonal↗