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Biomedical subjects

S Cavari

Publications and source records attributed to S Cavari.

7 recordsLinked to original sources
PubMed1996

Detection of heparin-like glycosaminoglycans in normal human plasma by polyacrylamide-gel electrophoresis.

This paper describes a procedure that allows detection of endogenous heparin present in normal human plasma by polyacrylamide-gel electrophoresis (PAGE). Plasma was submitted to proteolysis: an antithrombin III-dependent and heparinase I-sensitive anticoagulant activity was demonstrated in the supernatant of the digest. The supernatant was submitted to sequential fractionation with increasing concentrations of ethanol (25%, 50%, 60% and 65%, by vol.). Fractions were analyzed by PAGE for both glycosaminoglycan (GAG) and protein content. GAGs were detected by gradient PAGE (24-30%). The fraction obtained by 60% ethanol precipitation contained heparinase I-sensitive GAG. We show that GAGs co-precipitate with proteins. The SDS-PAGE of the material resulting from proteolytic digestion and subsequent ethanol fractionation, revealed three major bands. These peptides co-precipitated with plasma GAGs, mainly with the fraction obtained by 60% ethanol. We discuss the possibility that circulating endogenous heparin interacts with such peptides.

Electrophoresis, Cellulose Acetate↗
PubMed1996

Opposite effects of basic fibroblast growth factor and heparin on heparin/heparan sulfate degrading enzymes from Flavobacterium heparinum.

Heparinase I is normally inhibited by an excess or substrate, in this paper we describe that bFGF counteracts the inhibiting effect of the substrate and that heparinase I activity is increased in the presence of bFGF. This effect was observed using heparin concentrations ranging from 10(-9) to 10(-7) M with either a ten fold molar excess or an equimolar concentration of bFGF. In addition, the degree of depolymerization of heparan sulfate produced by heparitinase from Flavobacterium heparinum was increased in the presence of bFGF.

Chromatography, Gel↗
PubMed1994

Binding and growth-inhibitory effect of heparin and oligo-heparin (2kDa) in Balb/c 3T3 cells: lack of effect on PDGF- or serum-induced inositol lipid turnover.

1. The ability of heparins (bovine heparin sm 1026, Av. mol. wt. 36.9 kDa and bovine heparin EP 756, Av. mol. wt. 12.9 kDa) and heparin fractions of different molecular weights (low molecular weight heparin, LMW 2123/OP, Av. mol. wt. 4.5 kDa and oligo-heparin, Av. mol. wt. 2 kDa) to inhibit the proliferation and signalling of Balb/c 3T3 fibroblasts was investigated. 2. Heparin and heparin fractions of 4.5 and 2 kDa significantly inhibited DNA synthesis as monitored by [2H]-thymidine incorporation. 3. 3H-labelled heparin fractions of 4.5 and 2 kDa were prepared by gel-chromatography fractionation on Sephadex G-75 of an 3H-labelled commercial heparin after treatment with heparinase. 4. The binding of unfractionated and oligo-heparin of 2 kDa to Balb/c 3T3 fibroblasts was studied; we determined the specificity of heparin and oligo-heparin binding to the cells by means of displacement of bound 3H-labelled compound in response to increasing concentrations of unlabelled compounds. Scatchard analysis of binding data obtained using [3H]-heparin as ligand revealed the presence of a single class of high affinity binding sites (Kd = 28 nM) for heparin. Scatchard analysis of binding data obtained using [3H]-oligo-heparin as ligand revealed the presence of a single class of low affinity binding sites (Kd = 3.2 microM) for oligo-heparin. 5. In addition heparin displaced [3H]-oligo-heparin at a concentration of approximately 100 fold of the Kd determined in displacement studies. Furthermore, oligo-heparin significantly displaced [3H]-heparin at a concentration of approximately 10 fold of the Kd determined by displacement studies. 6. Both heparin and oligo-heparin exert their inhibitory effects on Balb/c 3T3 DNA synthesis stimulated by PDGF or serum. However these molecules did not affect the inositol lipid turnover triggered by PDGF at a concentration which did not produce maximal response. The increase of inositol phosphate metabolism produced by 20% serum was also unaffected by heparin. This concentration of serum elicited a response comparable to that induced by a submaximal concentration of PDGF.

3T3 Cells↗
PubMed1993

Glycosaminoglycans exposed on the endothelial cell surface. Binding of heparin-like molecules derived from serum.

We have analyzed the glycosaminoglycans exposed on the surface of endothelial cells cultured in vitro. The cells were labelled with [35S]sodium sulfate. Chondroitin sulfate, heparan sulfate and dermatan sulfate are the main sulfated glycosaminoglycans present on the cell surface. They are synthesized by endothelial cells. However, the electrophoretic analysis and corresponding autoradiography of the glycosaminoglycans removed from the endothelial cell surface shows the presence of heparin-like compound which is not synthesized by the cells as it is unlabelled by [35S]sodium sulfate. We show here that a proteolytic treatment of the commonly used serum for cell culturing, reveals the presence of an heparin-like anticoagulant activity. These results suggest that the endothelial cells bind endogenous heparin-like molecules present in serum.

Animals↗
PubMed1993

Antiproliferative effects of heparin on normal and transformed NIH/3T3 fibroblasts.

We studied the effect of heparin on proliferation and signalling in normal NIH/3T3 fibroblasts, and in cells transformed by different oncogenes. Heparin inhibited the proliferation of normal as well as of v-sis and v-erbB transformed fibroblasts in the presence of serum, but failed to inhibit v-erbB-driven proliferation in serum-starved cultures; under these conditions, heparin inhibited by approximately 50% the proliferation of normal and v-sis- transformed cells. Heparin also inhibited PDGF-induced cell proliferation and inositol lipid turnover in v-sis transformants, but it did not affect PDGF mitogenic signalling in NIH/3T3 fibroblasts.

3T3 Cells↗
PubMed1992

Endogenous heparinase-sensitive anticoagulant activity in human plasma.

In this paper we show that an anticoagulant activity, which we measure by thrombin time, appears in human plasma after its exhaustive proteolytic digestion. This activity is extremely heat stable, it is resistant to chondroitin ABC lyase (E.C.4.2.2.4) and heparan sulfate lyase (E.C.4.2.2.8), it is sensitive to heparin lyase (E.C.4.2.2.7) and to nitrous acid treatment: we suggest that it can be identified as authentic heparin. The amount present in 1 ml of plasma of healthy subjects corresponds to 0.1-0.2 I.U. of standard heparin (150 I.U./mg). Proteolytically digested human plasma was submitted to ion-exchange chromatography on DEAE-Sephacel and the anticoagulant activity in the fractions eluted at the different molarities of NaCl was measured by thrombin time. This analysis shows that the anticoagulant activity elutes at very low ionic strength. The possibility that interactions of the endogenous heparin with proteins or protein fragments are responsible for the difficulty in isolating heparin from human plasma is discussed.

Chromatography, Ion Exchange↗