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L Rosenfeld

Publications and source records attributed to L Rosenfeld.

At least 19 recordsLinked to original sources
PubMed1992

Heparin enhances active site-dependent binding of tissue-type plasminogen activator to endothelial cells.

Human umbilical vein endothelial cells (HUVEC) in culture express two classes of binding sites for tissue-type plasminogen activator (t-PA). The high-affinity binding site has been identified as PA inhibitor type 1 (PAI-1), which binds to the catalytic portion of the molecule, while the second site binds t-PA through an active-site independent domain. Because recombinant t-PA (rt-PA) is often administered concomitantly with heparin, we investigated the effects of heparin on rt-PA binding to HUVEC. Preincubation of HUVEC with heparin at 4 degrees C increased the binding of radiolabeled rt-PA in a time- and dose-dependent manner. One-half maximal increase in binding was observed within 10 minutes of heparin addition. When HUVEC were preincubated with optimal concentrations (5 U/mL) of heparin for 4 hours at 4 degrees C, a 2.5- +/- 0.2-fold increase in specific binding was observed (mean +/- SEM, n = 12, P less than .01). Other highly sulfated glycosaminoglycans and fucoidan (a sulfated polymer of fucose) stimulated rt-PA binding as well, whereas glycosaminoglycans with lower sulfate content than heparin did not. Several results suggested that heparin increased the binding of rt-PA to "cell-associated" PAI-1. First, only active-site-dependent binding was enhanced by heparin, whereas binding of active-site blocked rt-PA was not affected. Second, extracts from HUVEC preincubated with heparin contained increased amounts of rt-PA-PAI-1 complexes as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Third, antibodies to PAI-1 blocked the increased binding entirely. HUVEC preincubated with heparin also bound increased amounts of enzymatically active radiolabeled urokinase-type PAs. However, HUVEC preincubated with heparin did not express increased amounts of immunoreactive PAI-1. Therefore, heparin, at therapeutic concentrations, may enhance or stabilize the association of PAs with endothelial cell-associated PAI-1.

Binding Sites↗
PubMed1991

Comparison of the separation of bovine heparin by strong anion exchange and by gel filtration chromatography.

Heparin has been fractionated by strong anion exchange chromatography followed by elution of the pools on a gel filtration column. This resulted in the expected inverse relationship in the elution order for the pools run by the two methods. Also chromatography of heparin was performed in the reverse order: gel filtration first, followed by anion exchange of the pools. For this order of separation four of the five gel filtration pools of different molecular weights eluted at a similar LiCl concentration. The specific activities of different pools of heparin were evaluated using a colorimetric microwell kinetics assay using antithrombin and thrombin. For the pools separated by ion exchange first, there was an exponential increase of specific activity with increasing molecular weight for all pools. For the pools isolated by gel filtration first, the specific activities became level after an initial increase in relation to molecular weight. Thus, unique pools of heparin species are being isolated by different modes of chromatography.

Animals↗
PubMed1991

Interaction of fibronectin with heparin in model extracellular matrices: role of arginine residues and sulfate groups.

The interaction of heparin with the NH2-terminal domain of human plasma fibronectin was studied by using matrix-driven translocation, an assay for the adhesion of extracellular macromolecules with cell or particle surfaces within artificial collagen matrices. Partial desulfation of heparin rendered it ineffective in competitively inhibiting the interaction of the fibronectin NH2-terminal domain with heparin-coated particles, suggesting a role for sulfate groups of heparin in the interaction. Analysis of the fibronectin domain in terms of its primary structure, its proposed organization into "type I modules", and its hydrophilic and flexible segments led to the identification of several arginine-containing sites of potential interaction with the sulfate groups of heparin. Modification of increasing numbers of arginine side chains with 1,2-cyclohexanedione under mild conditions eventually led to decreases in translocation-promoting activity, and of heparin binding capacity as measured in a gel-shift assay, but the major portions of these functions were retained even when the four most accessible arginines (attributed to sites in and adjacent to the large loops of the type I modules) were modified. With the modification of additional arginines (attributed to sites in the small loops), both functions were lost. The peptide Gly-Arg-Gly, corresponding to a repeated determinant at the tips of two small loops, inhibited translocation, but arginine alone did not. Cleavage of the large loops by CNBr also led to loss of translocation-promoting activity. The correspondence between the molecular determinants of matrix-driven translocation and those previously found for mesenchymal morphogenesis indicates the utility of this system in the analysis of adhesive interactions of biological importance.

Amino Acid Sequence↗
PubMed1990

Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA.

Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC lead to an increase in its apparent molecular mass by approximately 40 Kd. The predominant u-PA binding protein isolated from whole cell detergent extracts migrated with a molecular mass of approximately 36 Kd using affinity chromatography. In contrast, when only cell surface proteins were radiolabeled before extraction, the predominant labeled u-PA binding protein isolated migrated with a molecular mass of approximately 46 Kd. Several pieces of evidence suggested that the difference in molecular mass between these two u-PA binding proteins resulted from glycosylation of a single receptor protein. First, a polyclonal antibody against u-PA receptor isolated from phorbol myristate acetate (PMA) stimulated U-937 cells reacted with both the 36- and 46-Kd proteins on Western blotting. Second, the size of the unmodified receptor was estimated by amplifying a full-length cDNA for u-PA receptor from an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence of a single 1.4-kilobase (Kb) mRNA transcript on Northern blot analysis predict an unglycosylated receptor protein of approximately 35 Kd. Third, synthesis of 35S-labeled 46-Kd cell surface receptor protein was inhibited when the cells were grown in the presence of tunicamycin, while the synthesis of the 36-Kd species was unaffected. Moreover, the apparent molecular mass of purified surface-labeled receptor (approximately 46 Kd) was reduced by N-glycanase. These studies suggest that the u-PA receptor on the surface of HUVEC is a glycoprotein derived from a protein of approximately 35 Kd which is similar immunologically to u-PA receptors on other cell types.

Base Sequence↗
PubMed1990

Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells.

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.

Binding, Competitive↗
PubMed1989

Lipoprotein analysis. Early methods in the diagnosis of atherosclerosis.

Before 1950, there was no clear perception of the interrelationship of serum lipids, atherosclerosis, and coronary heart disease. Since then, research laboratories have made conflicting claims for the most useful measurement of the serum lipid levels in detecting and managing coronary heart disease. Emphasis has been placed in turn on the measurement of levels of serum cholesterol, lipoproteins, triglycerides, and, currently, cholesterol and lipoproteins again. Physical separation and characterization of serum lipoproteins by ultracentrifugation and electrophoresis resulted in two classification systems for lipoproteins based on hydrated density and electrophoretic mobility, respectively. Two operational by-products were the atherogenic index, an empirical formula supposed to correlate with coronary heart disease, and a phenotype system for classification of the lipoproteinemias. Current National Heart, Lung, and Blood Institute criteria for atherosclerosis risk implicate elevated levels of cholesterol and low-density lipoprotein cholesterol, and decreased levels of high-density lipoprotein cholesterol. Although triglycerides are closely associated with cholesterol in lipoprotein molecules and are positively associated with cardiovascular disease, there is no strong evidence of elevated levels of plasma triglycerides as an independent risk factor in coronary heart disease. Elevated levels of triglycerides can help identify persons with increased risk of cardiovascular disease from other causes, but screening for hypertriglyceridemia is not recommended. Apolipoproteins and lipoprotein Lp(a) are briefly discussed.

Arteriosclerosis↗
PubMed1989

Hodgkin's disease: origin of an eponym--and one that got away.

Hodgkin's disease, the syndrome of enlarged lymph nodes associated with enlarged spleen, was described in 1832 on the basis of the gross appearance of the affected parts. The eponym entered the medical literature after repeated attempts by Dr. Samuel Wilks to credit Thomas Hodgkin with the priority of discovery. Today, this malady, also known by many other names, is recognized by microscopic examination of histological features. Hodgkin also described retroversion of the aortic valves with insufficiency in 1827 but Dr. Dominic Corrigan, whose report appeared in 1832, was credited with the eponym, despite efforts again by Wilks on behalf of Hodgkin.

History, 18th Century↗
PubMed1989

Martin Robison Delany (1812-1885): physician, black separatist, explorer, soldier.

Martin Robison Delany's great contribution to American life and black history stems from his defiant blackness. He was America's first "Black Nationalist" and the intense embodiment of black pride. Ever changing in his career, he always identified with the black experience and its place in history. In an active life he was doctor, dentist, orator, editor, publisher, Harvard medical student, explorer, dabbler in Central American politics, army officer, and Reconstruction office seeker.

Black or African American↗
PubMed1988

Location of specific oligosaccharides in heparin in terms of their distance from the protein linkage region in the native proteoglycan.

Studies were conducted to define the location of components and sequences in heparin with respect to their distance from the peptide linkage in the native proteoglycan. A purified heparin-oligopeptide was linked via its amino terminus to a matrix containing an azo bond and an activated carboxyl group. The polysaccharide chain was maximally degraded, either with heparinase or nitrous acid, and the soluble products were removed. The heparin-oligopeptide fragments that remained on the matrix were released by reductive cleavage of the azo linkage and characterized. The fragments, as well as heparin released without prior degradation, contained serine and glycine as the principal amino acids; the ratio of galactose to xylose was 2:1. The ratio of glucosamine to serine of 33:1 in the undegraded heparin was reduced to 6:1 and 1:1 in the heparinase-treated and nitrous acid-treated products, respectively. The undegraded sample and the fragments contained phosphate in equivalent amounts, demonstrating its presence in the heparin-protein linkage region. The heparin-oligopeptide preparation was also fractionated by gel filtration and high and low molecular weight fractions thus obtained were each linked to the insoluble matrix. The products that were subsequently released were subfractionated on a molecular weight-calibrated column of Sephadex G-200, and eluates were assayed for activity in promoting the neutralization of thrombin and factor Xa by antithrombin. The results revealed a sharp decrease in specific activity in heparin-oligopeptide fractions below Mr = 15,000 indicating that the anticoagulant-conferring segment is located at about 20 disaccharide units away from the peptide linkage region.

Amino Acids↗
PubMed1986

A fragment of antithrombin that binds both heparin and thrombin.

In order to identify the regions of antithrombin that interact with heparin and thrombin, it was degraded with CNBr and the activities of the isolated products were investigated. These fragments did not exhibit direct thrombin-neutralizing activity; however, one unique fragment was found to bind to heparin-Sepharose and also to interfere with the inhibition of thrombin by intact antithrombin. This fragment was identified as the one consisting of three disulphide-linked polypeptide chains containing residues 1-17, 104-251 and 424-432. At a concentration of 46 nM, this product decreased the heparin-enhanced thrombin-inhibitory activity of antithrombin by half, and completely abolished this inhibition when above 300 nM. In the absence of heparin, the action of antithrombin was not completely nullified by the fragment, even when present at relatively high concentrations. At a given fragment concentration, the extent of inhibition was independent of antithrombin concentration over the range tested. It was found that the fragment decreased the second-order rate constant for the antithrombin-thrombin reaction. Reduction and alkylation of the fragment showed that the above properties reside primarily in the peptide with residues 104-251. It is concluded that this peptide possesses portions of the antithrombin molecule that bind to heparin as well as to a site on thrombin.

Alkylation↗
PubMed1986

Papillary cystic oncocytoma and Warthin's tumor of the parotid gland.

A 69-year-old white female had a left superficial parotidectomy for a papillary cystic oncocytoma. The histologic appearance was identical to Warthin's tumor except that it lacked a lymphoid component. This case suggests that the general morphology of a Warthin's tumor does not depend on the presence of lymphoid tissue or association with an intra-parotid lymph node. We reviewed 50 patients with Warthin's tumors to investigate the histogenesis of the lesion. We found that most Warthin's tumors (86%) appeared to be within lymph nodes, as indicated by the presence of a lymph node capsule or sinuses. The anatomy of intra-parotid lymph nodes in glands containing Warthin's tumors has been compared with that found in 11 parotid glands containing mucoepidermoid carcinomas. So-called heterotopic ductal inclusions may actually represent the normal intimate relationship of parotid gland to intra-parotid lymph nodes.

Adenolymphoma↗