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D D Wagner

Publications and source records attributed to D D Wagner.

124 records · Page 7Linked to original sources
PubMed1985

Inhibition of disulfide bonding of von Willebrand protein by monensin results in small, functionally defective multimers.

The biosynthesis of von Willebrand protein by human endothelial cells was impaired by the presence of the carboxylic ionophore monensin. Several processing steps that have been localized to the Golgi apparatus were affected in a dose-dependent manner, including carbohydrate processing, dimer multimerization, and precursor cleavage. Since multimerization was more susceptible to the ionophore than was precursor cleavage, it appears that these processing steps are separate events. As expected, dimer formation, which occurs in the rough endoplasmic reticulum, was unaffected by monensin. Thus, at high concentrations of monensin, only dimer molecules were produced and secreted. The observed inhibition of multimer formation and precursor cleavage were not likely the result of incomplete carbohydrate processing, since inhibition of complex carbohydrate formation by swainsonine did not interfere with the other processing steps. Monensin also affected the capacity of endothelial cells to store von Willebrand protein, as the ratio of secreted to cell-associated protein increased dramatically in the presence of monensin, and the processed forms could not be found in the treated cells. The low molecular weight multimers produced in the presence of monensin did not incorporate in the endothelial cells' extracellular matrix nor did they bind to the matrix of human foreskin fibroblasts. In summary, the presence of monensin in human endothelial cell culture produced experimental conditions that mimic Type IIA von Willebrand disease, in that the cells synthesized and secreted only low molecular weight von Willebrand protein multimers, which were functionally defective.

Alkaloids↗
PubMed1985

Biosynthesis of von Willebrand protein by human megakaryocytes.

Immunofluorescence staining of buffy coat smears from a patient with chronic myelogenous leukemia in accelerated phase showed that approximately 13% of all nucleated cells contained von Willebrand protein and, therefore, appeared to be of megakaryocytic origin. This was confirmed by positive staining with antisera against platelet factor 4 and platelet glycoproteins. Short-term cultures of the buffy coat, which lacked endothelial cells, were metabolically labeled with [35S]methionine, and von Willebrand protein was immunopurified from cell lysates and culture medium. Cultures from this patient synthesized and secreted von Willebrand protein, in contrast with cultures from other patients with leukemia, who lacked circulating megakaryocytes, and from normal volunteers. The subunit composition of the megakaryocytic von Willebrand protein was very similar to that of human umbilical vein endothelial cells. The size of the processed subunit (220 kD) and of the cellular (260 kD) and secreted (275 kD) precursors from the two cell types were indistinguishable by gel electrophoresis. Furthermore, the ratio of precursor to processed subunit and the pattern of cellular and secreted nonreduced multimers were very similar. It appears, therefore, that the processing steps in biosynthesis of von Willebrand protein used by the megakaryocytes are very similar to those of umbilical vein endothelial cells.

Adult↗
PubMed1984

Von Willebrand protein binds to extracellular matrices independently of collagen.

Von Willebrand protein is present in the extracellular matrix of endothelial cells where it codistributes with fibronectin and types IV and V collagen. Bacterial collagenase digestion of endothelial cells removed fibrillar collagen, but the pattern of fibronectin and of von Willebrand protein remained undisturbed. Exogenous von Willebrand protein bound to matrices of different cells, whether rich or poor in collagen. von Willebrand protein also decorated the matrix of cells grown in the presence of alpha, alpha'-dipyridyl, a collagen-secretion inhibitor. These results indicate that the presence of von Willebrand protein in the extracellular matrix of endothelial cells and the binding of von Willebrand protein to foreign matrices do not depend on the matrix collagen.

2,2'-Dipyridyl↗
PubMed1984

Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization.

Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer chains and the 260,000-mol-wt monomers dimerized by interchain disulfide bond formation. The other processing steps have been localized to the Golgi apparatus and later compartments (e.g., Weibel-Palade bodies). High mannose carbohydrate was converted to the complex type, leading to the appearance of a larger precursor subunit of 275,000 mol wt. The 275,000-mol-wt species was not formed if carbohydrate processing was inhibited by the ionophore monensin. From the large pool of dimers of precursor subunits, the high molecular weight multimers were built. These dimer molecules appeared to have free sulfhydryls which might have been involved in the interdimer disulfide bond formation. Simultaneously with multimerization, the precursor subunits were cleaved to the 220,000-mol-wt form. The cleavage of the pro-sequence was not likely to be an absolute requirement for von Willebrand protein multimerization or secretion, as the 275,000-mol-wt precursor subunit was present in secreted high molecular weight multimers of the protein.

Blood Coagulation Factors↗
PubMed1984

Irradiation induces release of von Willebrand protein from endothelial cells in culture.

Human umbilical vein endothelial cells in tissue culture were irradiated with doses between 0 and 40 Gy, and the released von Willebrand (vW) protein and that which remained associated with the cells was quantitated. Doses of 20 Gy and higher produced a statistically significant increase in amount of vW protein secreted. This release was present whether the cells were labeled continuously throughout the experiment or just prelabeled before irradiation. An increase in fibronectin secretion was not observed. The release response to radiation was slow, reaching significance close to 24 hours after irradiation. The release of vW protein was not due to cell lysis, because the secreted vW protein contained very little of the large 260-kilodalton vW precursor subunit present in cell lysates and the cells appeared intact by immunofluorescence staining.

Blood Coagulation Factors↗
PubMed1983

Biosynthesis of von Willebrand protein by human endothelial cells. Identification of a large precursor polypeptide chain.

von Willebrand protein subunit is first synthesized by human endothelial cells as a larger precursor of 260-kDa that is slowly cleaved to its 220-kDa form. Both the precursor and the finished product are glycosylated. Small amounts of uncleaved precursor are secreted into the culture media. Disulfide bonded polymers typical for von Willebrand protein are also formed inside the cells. In comparison to fibronectin the biosynthetic processing of von Willebrand protein is longer as a delay in secretion is observed. The presence of a higher cellular pool of von Willebrand protein as compared to fibronectin indicates a storage compartment for von Willebrand protein in the endothelial cells.

Blood Coagulation Factors↗
PubMed1983

Subchronic toxicity of monensin in broiler chickens.

One hundred ninety-two male broiler chicks were dosed with monensin at concentrations of 0, 121, or 242 mg/kg feed throughout the normal growing period (50 days). Body weight gain and feed efficiency were determined weekly, and cardiac muscle was examined grossly and histologically at the end of the experiment. Livers also were weighed and examined grossly. Feed intake was determined daily, allowing continuous monitoring of drug intake. No depressing effects of the drug on growth rate and efficiency were observed until after four weeks, and then were evident only in the chicks receiving the 242 mg/kg diet. Subepicardial hemorrhage and congestion occurred in 40% of the hearts from the chickens fed the high monensin dose and were nonexistent in the other treatments. There appeared to be an inverse relationship between monensin dose and liver weight. The paralytic effects previously reported from acute dosing experiments were not observed. The results show that the heart and probably the liver are sensitive indicators of monensin toxicity and that the subchronic toxic dose is less than 18 mg/kg body weight per day.

Animal Feed↗
PubMed1982

Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells.

Immunofluorescence staining of cultured human umbilical vein endothelial cells has shown the presence of von Willebrand protein in the perinuclear region, in small rodlike structures through the cytoplasm, and on filaments of the extracellular matrix. Nonendothelial cells showed no staining with anti-von Willebrand protein antiserum. At the light microscope level, immunoperoxidase treatment of endothelial cells revealed the same pattern and antibody specificity as the fluorescence staining. Thin sections of the peroxidase-stained cells showed decorated filaments close to the substratum and also specific deposits in the endoplasmic reticulum and Weibel-Palade bodies. Control antisera against other selected proteins in endothelial cells failed to stain the Weibel-Palade bodies. These data suggest that the Weibel-Palade bodies of endothelial cells are storage and/or processing organelles for von Willebrand protein.

Blood Coagulation Factors↗
PubMed1981

Similarities and differences between the fibronectins of normal and transformed hamster cells.

Fibronectins from normal and virally transformed hamster cells were compared by several criteria. The fibronectin from transformed cells was similar to that from normal cells in being an intact dimeric glycoprotein with the ability to bind to gelatin, activated thiol-Sepharose, and cells. No evidence was found for proteolytic cleavages or abnormalities in disulfide bonding of transformed cell fibronectin. This fibronectin was also shown to be active in promoting cell attachment, elongation, and alignment. Therefore, the fibronectin produced by transformed cells is not defective. However, it was shown that the transformed cells were partially deficient in their capacity to bind fibronectins from either normal or transformed cells. This deficiency has implications for the significance of the loss of fibronectin on oncogenic transformation. Partial proteolysis of the fibronectins from normal and transformed cells gave rise to the same fragments. However, the glycosylated fragments from transformed cell fibronectin appeared somewhat larger than those from normal cell fibronectin. Analysis of fibronectin glycopeptides showed that transformation leads both to more branches per core and to a higher sialylation of the asparagine-linked complex carbohydrate side chains.

Animals↗
PubMed1979

Domain structure of fibronectin and its relation to function. Disulfides and sulfhydryl groups.

Hamster cell fibronectin is a glycoprotein consisting of two 230,000-dalton subunits in a disulfide-bonded dimer. The molecule is composed of domains which can be separated by partial proteolytic cleavage. The carbohydrates, disulfide bonds, and a single free sulfhydryl group per chain are distributed nonuniformly among these regions. All the interchain disulfides are within 10,000 daltons of the end of the molecule and are removed by mild proteolysis which also generates 200,000- and 25,000-dalton fragments which do not contain interchain disulfides. The 200,000-dalton fragment contains all or most of the carbohydrate side chains, and the free sulfhydryl group, but is relatively poor in cystine. The 25,000-dalton fragment is carbohydrate-free and cystine-rich but has no free sulfhydryl groups. There is heterogeneity in carbohydrate content among the monomeric chains of intact fibronectin and the 200,000-dalton fragments. The gelatin binding site of fibronectin is in the 200,000 fragment. Intact disulfide bonds are required for binding of fibronectin to cells and to gelatin and blockage of the free sulfhydryl groups prevents binding of fibronectin to cells, suggesting that intermolecular disulfide bonding may be important.

Amino Acids↗
PubMed1979

Cell surface fibronectin and oncogenic transformation.

Fibronectin is a large glycoprotein at the cell surface of many different cell types; a related protein is present in plasma. Fibronectin is a dimer of 230,000-dalton subunits and also occurs in larger aggregates; it forms fibrillar networks at the cell surface, between cells and substrata and between adjacent cells, and it is not a typical membrane protein. Cell surface fibronectin is reduced in amount or absent on transformed cells and in many cases its loss correlates with acquisition of tumorigenicity and, in particular, metastatic ability. Exceptions to the correlations with transformation and tumorigenicity exist. Loss of fibronectin and the resulting reduced adhesion appear to be involved in pleiotropic alterations in cell behavior and may be responsible for several aspects of the transformed phenotype in vitro. Fibronectin interacts with other macromolecules (collagen/gelatin, fibrin/fibrinogen, proteoglycans) and is apparently connected to microfilaments inside the cell.

Animals↗