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I Danishefsky

Publications and source records attributed to I Danishefsky.

At least 19 recordsLinked to original sources
PubMed1996

Correlation of expression of connexin mRNA isoforms with degree of cellular differentiation.

Examination of rat hepatic cell lines has revealed a correlation between the differentiated state of the cells and the gap junctional proteins, or connexins, they express. The cell lines RLC (Gershenson et al, 1970) and FTO.2B (Killary et al, 1984) were examined and compared to primary adult hepatocytes for expression of fetal and adult hepatic antigens under various tissue culture conditions. Maximal expression of fetal antigens was observed in cells grown in serum-supplemented medium, on either tissue culture plastic or type IV collagen. Maximal expression of adult specific antigens was seen in cells grown in a hormonally defined medium containing heparin, on type I or type IV collagen. The cell line RLC strongly expressed fetal antigens, while FTO.2B expressed both fetal and adult antigens. These cell lines and another poorly differentiated hepatic cell line, WB-F344 (Tsao et al., 1984) were used to assess the developmental profile of mRNAs encoding isoforms of gap junctions: connexins 26, 32, and 43. The cell lines each transcribed mRNAs of all three connexins, as determined by transcriptional elongation analysis. By contrast, only certain of the connexin mRNAs could be detected in specific cell lines by Northern analysis: RLC expressed only connexin 43 mRNA; WB-F344 expressed connexin 32 and 43 mRNAs. Selection among the connexin mRNAs appears to occur post-transcriptionally. Culture of the cell lines in hormonally defined medium vs. serum supplemented medium did not affect the patterns of connexin mRNA abundance. When the cell lines were cultured in hormonally defined medium containing heparin, however, the level of connexin mRNAs did vary: Connexin 26 mRNA increased in WB-F344 cells, and connexins 32 and 43 mRNAs increased in FTO.2B, but connexin 43 mRNA decreased in WB-F344 and RLC. The abundance of connexin mRNAs also varied when the cell lines were analyzed at different cell densities: connexin 43 mRNA increased with cell density in RLC and WB-F344, and connexin 26 mRNA peaked at an intermediate density and fell at higher cell densities in WB-F344. The differences in connexin mRNA expression among cell lines characteristic of different stages of hepatic differentiation, and the differences in regulation of connexin mRNAs in the hepatic cell lines, suggest distinct biological roles of the highly homologous proteins. Moreover, connexin gene expression may be a marker of hepatic development: as hepatocytes differentiate the proportions of connexin 43 then 26 mRNAs decrease while that of connexin 32 mRNA increases.

Animals↗
PubMed1994

Synthesis of proteoglycan during HL-60 cell differentiation.

Newly synthesized proteoglycans (PG) in proliferating HL-60 cells were labeled with [35S]-sulfate and fractionated on DEAE-cellulose columns. Peak fractions were digested with chondroitinase ABC and separated by paper chromatography. Chondroitin sulfate was identified as the major PG. Treatment of cells with 16 nM TPA resulted in a 1.8-fold increase in total sulfate incorporation into PG, a shift in its location from the cellular to the extracellular compartment, and an increase in the total charge of PG (based on the profile of elution from DEAE-cellulose columns), compared to controls. Incorporation of [3H]-glucosamine into cellular PG was markedly decreased in TPA-treated cells; the sulfate/glucosamine ratio showed a 13.5-fold decrease in the newly synthesized cellular PG. The sulfate/glucosamine ratio, however, was increased by 7.6-fold in secreted PG.

Cell Differentiation↗
PubMed1993

Changes in proteoglycan core protein (PCP) mRNA levels during HL-60 cell differentiation.

The steady-state level of the PCP mRNA was investigated during HL-60 cell differentiation. When cells were induced to differentiate into granulocytes with 1.3% dimethylsulfoxide or 1 microM cis or trans retinoic acid, no significant changes in PCP mRNA levels were observed. In contrast, cells cultured in the presence of the phorbol ester TPA, which promotes the cells to be differentiated into monocytes/macrophages, were associated with a marked decrease in the steady-state concentration of PCP mRNA. When cells were simultaneously treated with a combination of TPA and staurosporine, a protein kinase C (PKC) inhibitor, the TPA-elicited decrease in PCP mRNA was partially prevented. These data suggest that PCP mRNA expression is not directly linked to HL-60 cell differentiation but appears to involve the participation of PKC.

Aggrecans↗
PubMed1992

Depolymerization of heparin by complexed ferrous ions.

Treatment of porcine heparin with the ferrous-EDTA complex and ascorbic acid for 24 h at 37 degrees C results in the degradation of most of the glycosaminoglycan to smaller fragments. About 65% of the products comprise oligosaccharides composed of less than 30 sugar units. The extent of depolymerization is decreased significantly if ascorbate or EDTA is not included in the reaction mixture. Gel filtration of the reaction products yielded fractions with narrow chain length ranges. The sulfate content of the fractions and their electrophoretic mobilities on cellulose acetate indicate that the components have equivalent charge densities. Depolymerization products with 20 or more sugar units retain significant anticoagulant potencies as measured by their effect in accelerating the neutralization of factor Xa by antithrombin.

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↗
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

Requirement of free carboxyl groups for the anticoagulant activity of heparin.

The uronic acid carboxyl groups of a heparin fraction with high anticoagulant activity, were converted to the methyl ester by treatment with diazomethane. The product obtained after purification did not have the characteristic activity of heparin in accelerating the inhibition of thrombin or factor Xa, by antithrombin. Esterification also abolished the binding of heparin to antithrombin as measured by changes in the intrinsic fluorescence. It is concluded that free carboxyl groups are essential for the activity of heparin.

Anticoagulants↗
PubMed1985

Distribution of glucuronic and iduronic acid units in heparin chains.

The distribution of glucuronic and iduronic acid within the chains of anticoagulantly active and inactive beef lung heparin was investigated. A fraction with an average molecular weight of 19,500 was isolated from the heterodisperse mixture and then separated into active and inactive components by affinity chromatography. Each sample was linked through its reducing terminus to tyramine, reduced with sodium borotritide, and bound covalently to Sepharose via an azo bridge. The bound reduced heparin was treated with a limited amount of HNO2 and the degraded fragments were removed. The sections of the chain contiguous with the original reducing terminus were then detached from the insoluble matrix by reaction with sodium dithionite. The recovered polysaccharide was fractionated according to size on Sephadex G-200 and the amount of each uronic acid in the individual fractions was determined. Inactive heparin showed a constant percentage of glucuronic acid in all fragments, i.e. about 8.9% of the total uronic acid. With active heparin the percentage of glucuronic acid increased with the distance from the reducing terminus of the polysaccharide chain, ranging from 9.5 to 20% of the uronic acids. These results suggest that the biosynthesis of active heparin involves unique reactions or specific processing of the macromolecule.

Animals↗
PubMed1985

The uronic acid composition of anticoagulantly active and inactive heparin.

Bovine heparin was fractionated with respect to chain length and anticoagulant activity. Analysis of each of these fractions for iduronic and glucuronic acids demonstrated that active heparin has a greater amount of glucuronic acid than inactive heparin. The ratio of the uronic acids in the respective fractions was the same for heparin with different molecular weights. Thus, active heparin with longer chain lengths have more additional glucuronate residues than are required for the antithrombin-binding domain. The results indicate that the active and inactive heparin species differ in more than one structural characteristic and suggest a considerable divergence in their respective biosynthesis.

Animals↗
PubMed1984

Effects of enzymatic deglycosylation on the biological activities of human thrombin and antithrombin.

Sequential digestion of human thrombin and antithrombin with neuraminidase, beta-galactosidase, beta-N-acetylglucosaminidase, and endo-beta-N-acetylglucosaminidase D resulted in the successive removal of sialic acid, galactose, N-acetylglucosamine, and mannose and more N-acetylglucosamine residues. The products obtained after each stage of deglycosylation had electrophoretic mobilities that were consistent with the calculated change in mass expected from the cleavage of the sugar moieties. The modified thrombins did not lose fibrinogen-clotting activity, amidolytic activity, nor the ability to form complexes with antithrombin. In addition, asialothrombin and asialoagalactothrombin caused the same extent of platelet release as did control thrombin. The products obtained after removal of sugars from antithrombin retained thrombin-neutralizing activity. In the presence of heparin the inhibition of thrombin as well as factor Xa was enhanced. Thus, the sugar residues of thrombin and antithrombin are not required for the formation of enzyme-inhibitor complexes or for the other activities that were measured.

Acetylglucosaminidase↗
PubMed1984

Location on heparin of the oligosaccharide section essential for anticoagulant activity.

Studies were conducted to define the location of the unique oligosaccharide sequence in heparin that is required for its anticoagulant activity. A heparin fraction with an average molecular weight of 20,000 was linked at its reducing terminus to a Sepharose derivative via an azo bridge. The matrix-linked heparin was subjected to limited degradation with nitrous acid and, after the cleaved segments were removed, the section contiguous with the original reducing terminus was released from the gel by treatment with sodium dithionite. The results of analyses of the products after separation into different molecular weight pools demonstrate that the groups responsible for accelerating the neutralization of thrombin and Factor Xa by antithrombin are located at or near the nonreducing terminus of the heparin chain.

Animals↗
PubMed1983

Action of heparin on the inhibition of thrombin by alpha 1-proteinase inhibition.

alpha 1-Proteinase inhibitor inhibits the action of thrombin while heparin is effective in relieving this inhibition. Kinetic analyses of the inhibition in the absence and presence of heparin were conducted by employing the p-nitroanilide of a synthetic peptide, S-2238, to determine residual thrombin activity. Under pseudo-first-order conditions, the rate of thrombin neutralization was proportional to alpha 1-proteinase inhibitor concentration up to the highest concentration of inhibitor employed, 94.6 microM. The second-order rate constant under the conditions studied was 6.64 X 10(3) M-1 min-1, and heparin was found to cause a decrease in the rate constant. On the basis of the concentrations used, the kinetics of thrombin inhibition, both in the absence and in the presence of heparin, can be described by a one-step reaction. The effect of heparin is due to its binding to thrombin. The degree to which heparin interferes with the rate of thrombin inhibition depends on its molecular weight but not on its anticoagulant activity. Thus, in the presence of 11.7K and 22K heparins the respective rate constants are 3.5 X 10 and 1.0 X 10(3) M-1 min-1. Additionally, the concentration of heparin required to produce a maximal decrease in reaction rate depends on the molecular weight of the heparin employed.

Animals↗
PubMed1983

Applications of high-performance liquid chromatography to the characterization of the interactions of heparin with antithrombin III and thrombin.

A rapid method for differentiating active heparin from inactive components by high-performance liquid chromatography is described. Additionally, the procedure allows for the distinction of heparin fractions with different molecular weights. The methodologies were extended to investigations on the effects of heparin in the interaction between thrombin and antithrombin III. The product formed in the presence of heparin has a characteristic retention time that is ascribed to the binding of heparin to the protein-protein complex.

Animals↗