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Biomedical subjects

D F Mosher

Publications and source records attributed to D F Mosher.

At least 91 records · Page 5Linked to original sources
PubMed1992

Effects of sized heparin oligosaccharide on the interactions of Chinese hamster ovary cell with thrombospondin.

Binding and degradation of TSP by CHO cells and adhesion of CHO cells to substrate-adsorbed TSP are mediated by cell surface PGs and inhibitable by heparin. In order to learn how these three processes are related, we studied the effects of defined heparin oligosaccharides up to 18-mer produced by nitrous acid digestion. There was a complex correlation among oligosaccharide chain length, affinity of oligosaccharide for TSP in a solid phase binding assay, and potencies of oligosaccharide in inhibition of the three cellular processes. Inhibition of degradation was more sensitive to shorter oligosaccharides than inhibition of binding. For instance, the 10-mer inhibited binding of TSP to cells by 10% and degradation by 70%. Punctate immunofluorescence of cell surface bound TSP was replaced by a diffuse pattern after incubation in the presence of the 10-mer. These results suggest that the clustering of TSP on the cell surface may trigger endocytosis and degradation. Inhibition of binding of TSP to cells, in turn, was more sensitive to midsized oligosaccharides than inhibition of cell adhesion to adsorbed TSP. Inhibition of adhesion correlated with the ability of oligosaccharides to block binding of 125I-heparin to adsorbed TSP.

Adsorption↗
PubMed1992

Inhibition of cell adhesion by high molecular weight kininogen.

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.

Cations, Divalent↗
PubMed1992

Disulfides modulate RGD-inhibitable cell adhesive activity of thrombospondin.

Thrombospondin (TSP) contains the Arg-Gly-Asp (RGD) sequence that is thought to be important for cell adhesion mediated by several cell-surface integrin receptors. The RGD sequence is located in the type 3 repeat region of TSP that has multiple Ca2+ binding sites and is subject to a complex intramolecular thiol-disulfide isomerization. TSP that we isolated from thrombin-activated human platelets using buffers containing 0.1 mM Ca2+, in which Cys974 is the major labeled cysteine, did not have RGD-inhibitable adhesive activity. However, one of our preparations of TSP and TSP purified following alternative procedures using greater than or equal to 0.3 mM Ca2+ did have RGD-inhibitable adhesive activity. Reduction of TSP with DTT, either before or after adsorption to surfaces, enhanced its adhesive activity. Reduced TSP supported robust cell spreading when coated at concentrations as low as 1 micrograms/ml, whereas "adhesive" TSP not treated with DTT was active at coating concentration of greater than 20 micrograms/ml and supported only modest cell spreading. Lower DTT concentrations were required for enhancement of the adhesive activity of TSP if Ca2+ was chelated with EDTA. Cellular adhesion to DTT-treated TSP was inhibited by RGD-containing peptide and by mAb to a functional site of the alpha v beta 3 integrin. Cell blots of reduced proteolytic fragments of TSP localized the adhesive activity to the RGD-containing type 3 repeat region. These results suggest a novel mechanism for regulation of integrin-ligand interactions in which the ligand can isomerize between inactive and active forms.

Amino Acid Sequence↗
PubMed1992

Lipoproteins enhance fibronectin binding to adherent cells.

We have identified very low density (VLDL) and low density (LDL) lipoproteins as blood plasma components that enhance the binding and deposition of fibronectin into the extracellular matrices of cultured MG-63 osteosarcoma cells and human fibroblasts. The lipoproteins increased the binding and deposition of iodinated fibronectin by MG-63 cells threefold over control levels. LDL also increased the deposition of multimeric fibronectin into extracellular matrix as assessed by gel electrophoresis and fluorescence microscopy. High density lipoprotein (HDL) and the d > 1.21 g/ml nonlipoprotein fraction had less activity. Enhancement of binding of fibronectin was observed within 15 minutes, when binding was largely reversible. LDL also increased the binding of a fragment containing the 70-kd amino-terminal region of fibronectin that is primarily responsible for the reversible binding of fibronectin to cell layers. LDL had to be present simultaneously with radiolabeled fibronectin to exert an effect on fibronectin binding. LDL enhanced fibronectin binding equally well to normal skin fibroblasts and to familial hypercholesterolemic fibroblasts lacking the LDL receptor. Acetylation of LDL, performed to block its interaction with the LDL receptor, did not diminish the enhancement of fibronectin binding to MG-63 cells. These results indicate that LDL and VLDL interact with fibronectin to potentiate binding to monolayer cells through a pathway that does not involve the LDL receptor.

Amino Acid Sequence↗
PubMed1991

Five type I modules of fibronectin form a functional unit that binds to fibroblasts and Staphylococcus aureus.

Fibronectin is a cell-adhesive protein comprised of three types of repeating homologous sequences, I, II, and III (Petersen, T.E., Thøgersen, H.C., Skorstengaard, K., Vibe-Pedersen, K., Sahl, P., Sottrup-Jensen, L., and Magnusson, S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 137-141). The amino-terminal portion of fibronectin is comprised of five type I modules and mediates assembly of dimeric soluble fibronectin into insoluble fibrils by cultured fibroblasts, binding and cross-linking of fibronectin to Staphylococcus aureus, and binding and cross-linking of fibronectin to fibrin. It is not known whether these binding activities require individual type I modules, several modules, or all five modules. To answer this question, we generated recombinant truncated fibronectin molecules with deletions of or mutations in the amino-terminal type I modules. Binding to cellular fibronectin assembly sites and S. aureus required all five type I modules. In contrast, proteins with deletions of type I modules interacted well with fibrin.

Base Sequence↗
PubMed1991

Synthesis of truncated amino-terminal trimers of thrombospondin.

Thrombospondin (TSP) is a 450-kDa glycoprotein that is comprised of three identical disulfide-bonded subunits (1152 amino acids) held together near the heparin-binding amino-terminal globular domains. TSP truncated at residue 277 (TSP-277) or 381 (TSP-381) consisted largely of disulfide-bonded trimers when expressed in COS cells or insect cells. In addition, TSP-381 formed heterotrimers with endogenous COS cell TSP. Cleavage of TSP and the truncated mutants in the proteolytically sensitive region between residues 220 and 237 yielded monomeric amino-terminal fragments. Cys-252 and Cys-256 are the only cysteines between residues 238 and 277 and therefore must bridge among subunits. TSP-381 in which Cys-252 and Cys-256 were changed to glycine was secreted efficiently by COS cells but with only a minor portion of the protein in the form of disulfide-bonded trimers. The sequence of TSP between residues 258 and 283 is predicted to form an amphiphatic alpha-helix. We suggest that assembly of TSP trimers involves formation of an alpha-helical coiled-coil structure which is stabilized by formation of disulfides.

Amino Acid Sequence↗
PubMed1991

Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix.

Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III-1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin.

Antibodies, Monoclonal↗
PubMed1991

Assembly of fibronectin into extracellular matrix.

The results summarized above suggest that assembly of fibronectin is a fundamental biological process and that knowledge of the process of assembly may reveal new ways by which cells interact with extracellular molecules. Deposition of a fibronectin matrix seems to be regulated as tightly as synthesis of fibronectin or expression of adhesion receptors for fibronectin and is influenced profoundly by two products of blood coagulation--TGF-beta released from platelets and factor XIII activated by thrombin. Fibronectin assembly may be important in all sorts of physiological and pathophysiological processes. Cell A--for instance, a stromal cell--can influence the behavior of cell B--for instance, a lymphocyte--by assembling fibronectin made by cell C--for instance, a hepatocyte. We hope that the testable models of assembly presented in this paper will lead to new understanding of the process of assembly and suggest new modalities for treatment of diseases that result in fibrosis, damaged tissues, and neoplastic growth.

Amino Acid Sequence↗
PubMed1991

Vitronectin in bronchoalveolar lavage fluid is increased in patients with interstitial lung disease.

Vitronectin, also known as S-protein, is a 75,000-dalton serum glycoprotein that has a variety of functions, including the capacity to interact with the terminal components of the complement cascade, the coagulation system, and cell surfaces. By virtue of its ability to interact with cells, vitronectin is capable of mediating cell-spreading and adhesion and may also influence cell differentiation and cell growth. To investigate the possibility that vitronectin might contribute to the pathogenesis of interstitial lung disease, vitronectin was measured in bronchoalveolar lavage fluid from patients with sarcoidosis, idiopathic pulmonary fibrosis, and, for comparison, normal volunteers. Vitronectin was detected in lavage fluid and serum of all study subjects. Increased lavage fluid concentrations were found in patients with interstitial lung disease when compared with normal subjects (p less than 0.005), and glucocorticoid-treated patients with interstitial lung disease had lower vitronectin levels than did untreated patients. Furthermore, on SDS-PAGE and Western blot analysis lavage fluid vitronectin comigrated with serum vitronectin, suggesting similar molecular size. Thus, vitronectin is a normal constituent of the epithelial lining fluid, and lavage fluid vitronectin is similar to serum vitronectin. The increase of vitronectin concentrations in the epithelial lining fluid of patients with interstitial lung disease suggests that vitronectin may contribute to the pathogenesis of interstitial lung disease.

Blood Proteins↗
PubMed1991

Ca2(+)-sensitive binding of thrombospondin to U937 cells is due to the formation of calcium precipitate in the binding medium.

Thrombospondin (TSP) binds to U937 monocytic cells in a Ca2(+)-enhancible and EDTA-inhibitable manner (Silverstein, R. L., and R. L. Nachman. 1987. J. Clin. Invest. 79:867-874; Silverstein, R. L., A. S. Asch, and R. L. Nachman. 1989. J. Clin. Invest. 84:546-552). We reproduced the results when RPMI cell culture medium, but not when HBSS was used as binding medium. Addition of 1 mM Ca2+ to RPMI medium increased the binding of TSP to suspended U937 cells more than eightfold; the increase was blocked by EDTA but not by heparin. Further studies showed that addition of 1 mM Ca2+ to RPMI medium resulted in an insoluble precipitate, which did not form when EDTA was present or when 1 mM extra Ca2+ was added to HBSS. TSP bound to the precipitate in a saturable and specific manner. The precipitate enhanced binding of TSP to MG63 osteosarcoma cells in a monolayer binding assay. Enhancement of binding in the monolayer assay was observed for fibronectin and vitronectin as well. Our data indicate that there is not a specific Ca2(+)-dependent TSP receptor on U937 cell surface. Instead, the extra binding enhanced by Ca2+ is due to the formation of insoluble salts in the medium.

Binding Sites↗
PubMed1991

Vitronectin.

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Amino Acid Sequence↗
PubMed1990

Acute tumor lysis syndrome and treatment response in patients treated for refractory chronic lymphocytic leukemia with short-course, high-dose cytosine arabinoside, cisplatin, and etoposide.

Acute tumor lysis syndrome resulting from rapid neoplastic cell lysis after chemotherapy is an unusual event, generally seen in histological aggressive lymphatic tumors. The authors saw four patients who developed acute tumor lysis syndrome when treated for advanced-stage, refractory chronic lymphocytic leukemia (CLL) with an initial cycle of cytosine arabinoside (Ara-C) 2 g/m2 every 12 hours x 4, cisplatin 35 mg/m2 every 24 hours x 2, and etoposide 100 mg/m2 every 24 hours x 2 (ACE). With aggressive hydration, urine alkalinization, forced diuresis, and high-dose allopurinol, acute tumor lysis syndrome was not seen in three subsequent cases of CLL treated with ACE. Of a total of eight patients treated, seven patients had marked reductions in lymphocyte counts after the first course of ACE. Of the eight patients, three are alive: one in a complete remission greater than 2 years, one in partial remission after three cycles of ACE, and one in Richter's transformation to large cell lymphoma. The remaining patients died after one cycle of ACE chemotherapy, one as a direct complication of acute tumor lysis and pancytopenia, and four others from complications of severe pancytopenia and general debilitation. Therefore, ACE appears to cause a rapid dissolution of tumor cells in CLL, and with appropriate aggressive management of the tumor lysis and infectious complications may have a favorable impact on survival in advanced CLL.

Aged↗
PubMed1990

Binding and degradation of blood coagulation factor XIII by cultured fibroblasts.

The interaction of Factor XIII with cultured fibroblasts was examined using 125I-labeled protein and immunofluorescence. Platelet or plasma Factor XIII bound to confluent cell layers. Binding reached an apparent steady state after 8 h. Activation with thrombin increased the binding of both the platelet and plasma forms of the enzyme. After a 1-2 h lag, a chloroquine-inhibitable increase in trichloroacetic acid-soluble radioactivity was detected in the medium. Gel electrophoresis in sodium dodecyl sulfate indicated that approximately 16-fold more a subunit (catalytic) of 125I-plasma Factor XIII bound to the cell layer than b subunit (carrier) and that some large complexes containing Factor XIII were formed with the cell layer. Factor XIII binding increased linearly with concentrations of Factor XIII up to 230 micrograms/ml, whereas a component of the degradation of Factor XIII was saturable at about 20 micrograms/ml. Factor XIII associated with cell layers was catalytically active since it could cross-link fibronectin. By immunofluorescence the a subunit of Factor XIII was localized to fibronectin-containing extracellular fibrils and, in the presence of chloroquine, to intracellular granules. These results indicate that the a subunit of Factor XIII binds to the fibroblast extracellular matrix and matrix assembly sites, where it remains active, and to a putative cell-surface receptor which mediates its internalization and degradation.

Blood Platelets↗
PubMed1990

Recombinant 70-kDa protein from the amino-terminal region of rat fibronectin inhibits binding of fibronectin to cells and bacteria.

Binding of fibronectin to substrate-attached cells and to Staphylococcus aureus is mediated by the amino-terminal 70-kDa portion of fibronectin. The 70-kDa amino-terminus is composed of nine type I and two type II internal homology units, each containing two intrachain disulfide bonds. The exact structural features of the 70-kDa amino-terminus that are necessary for binding to cells and bacteria are not known. We characterized a recombinant 70-kDa protein from the amino-terminus of rat fibronectin using a baculovirus expression system. Recombinant 70-kDa (r70kDa) protein was easily purified in high amounts from the conditioned medium by affinity chromatography on gelatin-agarose. Secretion was much less when N-linked glycosylation was blocked by tunicamycin. Like the native fragment, the r70kDa protein contains intrachain disulfide bonds. In addition, the r70kDa protein was indistinguishable from the nonrecombinant 70-kDa fragment in its ability to compete for binding sites on fibroblasts and S. aureus. Thus, the r70kDa protein retains the important functional characteristics of the native fragment. This expression system is well adapted to studying the structural features important for the interaction of 70-kDa protein with cells.

Animals↗
PubMed1990

Co-assembly of plasma and cellular fibronectins into fibrils in human fibroblast cultures.

Exogenous plasma and endogenous cellular fibronectins on the surface of cultured fibroblasts and in extracellular matrix fibrils were colocalized by fluorescent and high voltage immunoelectron microscopy. Fibroblast cultures grown in the presence or absence of cycloheximide were incubated with exogenous plasma fibronectin labeled with fluorescein isothiocyanate. A monoclonal antibody specific for the EIIIA sequence of cellular fibronectin was used to detect cellular fibronectin. A rabbit antifluorescein antibody identified fluoresceinated plasma fibronectin. In cultures incubated in the presence of cycloheximide, plasma fibronectin was bound to the cell surface and was assembled into extracellular fibrils. In cultures grown in the absence of cycloheximide, plasma and cellular fibronectins were observed in the same matrix fibrils and in the same locations on the cell surface. There was not, however, random admixture of the two proteins.

Cell Membrane↗