Dextran Sulfate Concentration Research Articles

The surface-dependent autoactivation mechanism of factor XII.

The dependency of concentrations of Zn2+ and the negatively charged surfaces, phosphatidylinositol phosphate (PtdInsP), sulfatide and dextran sulfate, on the autoactivation of human factor XII, has been studied. While the autoactivation induced by sulfatide, and low concentrations of dextran sulfate, was unaffected by the presence of Zn2+, that induced by PtdInsP and higher concentrations of dextran sulfate was completely dependent on Zn2+: the excess of Zn2+ needed to induce maximal activity with PtdInsP was 12-fold the concentration of factor XII, while with dextran sulfate it was 40-fold. Determination of the Zn2+-binding properties of factor XII revealed that a total of four zinc ions could bind to each factor XII molecule. The first bound zinc ions (Kd 0.1 microM) induced an increase in the intrinsic tryptophan fluorescence of factor XII, while further titration up to a 40-fold surplus resulted in a quenching of the fluorescence. Binding of the zinc ions that caused the quenching had an average Kd of approximately 1 microM, independent of whether it was determined from the fluorescence changes or by equilibrium filtration. Low concentrations of both sulfatide and PtdInsP induced a fluorescence increase similar to that at low concentrations of Zn2+ but, in contrast to sulfatide, higher concentrations of PtdInsP did not induce a quenching in fluorescence. As the Zn2+-independent activating surface (sulfatide) induced quenching in the fluorescence intensity, while the Zn2+-dependent activating surface (PtdInsP) did not, the quenching, whether it was caused by sulfatide or zinc ions, was assigned to a change in the conformation which resulted in a molecular structure of factor XII that could be autoactivated. Association of factor XII in this conformation on the activating surface was suggested to be responsible for the autoactivation.

Surface modification of chitosan membranes by complexation-interpenetration of anionic polysaccharides for improved blood compatibility in hemodialysis

Chitosan membrane surface was modified by cornplexation and interpenetration of anionic polysaccharides - heparin and dextran sulfate - for improved blood compatibility in hemodialysis. Electron spectroscopy for chemical analysis results showed a characteristic sulfur (S) and sodium (Na) peaks after modification with dextran sulfate. The sulfur/carbon (S/C) atomic composition ratio increased from 0.03 to 0.08 when the bulk dextran sulfate concentration used for modification was increased from 2.5 to 10 mg ml-1 . The permeability of urea and creatinine did not change significantly upon modification with heparin or dextran sulfate. Surface modification, however, did decrease the permeability coefficients of glucose, vitamin B-2, and vitamin B-12. Unlike Cuprophan, chitosan and surface-modified chitosan membranes did not significantly activate the complement system as measured by the serum iC3b concentration. Compared to forty and sixty fully-activated platelets present on control surfaces, surface modification with heparin and dextran sulfate significantly reduced the number of adherent platelets per 25 000 μm2 area and the extent of platelet activation. Surface modification with anionic polysaccharides, however, did significantly shorten the plasma recalcification time leading to fibrin clot formation. The results of this study show that chitosan membrane surface can be modified by complexation-interpenetration of anionic modifying agents. The modified membranes do resist complement activation and platelet adhesion and activation.

Glomerular Processing of Dextran Sulfate during Transcapillary Transport

The fractional clearance of dextran sulfate across the glomerular capillary wall in the isolated perfused kidney is shown to be dependent on the concentration of dextran sulfate in the perfusate and the degree of sulfate substitution on dextran sulfate. While normal charge selectivity is apparent at low dextran sulfate perfusate concentrations, the nature of the concentration dependence of the fractional clearance clearly eliminates charge-electrostatic interactions as being responsible. A strong correlation has been experimentally established between fractional clearance and the degree of desulfation of dextran sulfate found in the urine. The correlation was further established through the demonstration that the lysosomotropic agent NH4Cl partially inhibits charge selectivity as well as desulfation. The glomerular cellular processing of the dextran sulfate was further studied through dextran sulfate-mediated release of metabolically labeled glomerular heparan sulfate. A model of glomerular capillary wall transport, invoking endothelial cell processing of dextran sulfate, is proposed to explain the differences between the fractional clearance of dextran and dextran sulfate.

Dextran sulphate induces a PKC and actin independent internalisation of CD4

Internalisation of CD4 is a well-known phenomenon. It occurs in the presence of phorbol myristate acetate (PMA), TPA or gangliosides and is usually completed within 30 min. Here, we describe an internalisation of CD4 molecules induced by low concentrations of high molecular weight dextran sulfate (M(r) 500 kDa) which differs from the classical mode in several ways. Internalisation is demonstrated by flow cytometry after simultaneous and consecutive staining of extracellular and internalised CD4 molecules and by visualisation by electron micrographs. A simple blockage of antibody binding sites on the CD4 molecule (epitope masking) by DS500, as widely believed, is definitely not responsible for the observed effects. DS500-mediated internalisation is a slow and energy-dependent process, where CD4 but not CD 2, 3, 8, 16, 56 and HLA-DR molecules are involved. The reaction reveals a characteristic time and concentration dependency. It does not require activation of protein kinase C (PKC) and cannot be inhibited by cytochalasin D. These results provide some more insight in the behavior of CD4 on lymphoid cell surfaces in response to high molecular weight polyanions such as dextran sulfate.

Enantiomeric separation of drugs by affinity electrokinetic chromatography using dextran sulfate.

Dextran sulfate (sodium salt), which is a mixture of a linear alpha-(1,6)-linked D-glucose polymer having a sulfate group in the molecule, has been employed as a chiral selector in affinity electrokinetic chromatography (EKC) for the separation of enantiomers of drugs. Enantiomers of trimetoquinol hydrochloride and its positional isomer have been successfully separated by the method using a 3-5% dextran sulfate buffer solution of pH 5.5 within 20 min. The effects of buffer pH, concentration of dextran sulfate, and additives (such as an organic solvent, urea and a surfactant) on the enantioselectivity were investigated. The choice of pH and the concentration of dextran sulfate has been important for the improvement in selectivity.

Detection System for Membrane Immunoassay Based on the Trapping of a Highly Colored Intermediate of the Peroxidase Reaction

The oxidation of ortho-dianisidine by membrane bound horseradish peroxidase in the presence of sodium dextran sulfate affords a dark-green insoluble product, identified as an unstable meriquinone intermediate previously reported in literature. Cationic and unsubstituted dextrans do not stabilize the intermediate. The highest yield of the intermediate is observed at pH 4.0-5.0 and concentration of dextran sulfate ca. 0.5%. New highly sensitive detection system for peroxidase has been developed on the basis of ortho-dianisidine oxidation to the meriquinone intermediate in the presence of sodium dextran sulfate. Under certain conditions, with lowered sodium dextran sulfate concentrations, a progressive further oxidation of the green intermediate to the yellow-brown final product is observed on passing to higher enzyme concentrations. This finding opens a possibility to develop a detection system in which the color of the mixture of reaction products serves as a measure of the enzyme concentration.

Inhibition of prekallikrein activation in human plasma by components of bovine plasma.

Contact of plasma with a negatively charged surface activates prekallikrein and factor XII reciprocally. Activation of prekallikrein by several activators was impaired in bovine plasma when compared to that in human plasma. The activated partial thromboplastin time of bovine plasma, induced by several activators, was significantly longer than that of human plasma. Cleavage of [125I]factor XII was optimum at 10 min in human plasma but took up to 60 min in bovine plasma. Addition of bovine plasma to human plasma caused significant inhibition of dextran sulfate-induced prekallikrein activation, indicating that the impaired rate of contact activation in bovine plasma is due to the presence of inhibitors. The inhibitory effect was greater at lower concentrations of dextran sulfate but could not be abolished by increasing the concentration. The inhibitory activity eluted in two peaks at low and medium salt concentrations on carboxymethyl ion-exchange chromatography of bovine plasma.

Dextran Sulfate Complexes with Potassium Phosphate to Interfere in Determinations of High-Density Lipoprotein Cholesterol

The interference of dextran sulfate with the colorimetric determination of free cholesterol causes an increase in the absorbance that is proportional to the dextran sulfate concentration in the sample. This makes estimation of the free cholesterol concentration unreliable in high-density lipoprotein-containing supernates obtained after plasma precipitation with dextran sulfate/MgCl2. Analysis of the absorption spectrum demonstrated that the absorbance increase was due to turbidity. We observed this effect with colorimetric reagents based on potassium phosphate buffer but not with those based on sodium phosphate or Tris buffers. This effect is ascribed to dextran sulfate because neither the omission of Mg2+ ions nor their chelation with EDTA prevented turbidity, whereas precipitation of dextran sulfate with Ba2+ counteracted this effect. Therefore, potassium phosphate-buffered reagents appear unsuitable for colorimetric lipid determinations in samples containing dextran sulfate.

Analysis of Intrinsic Fibrinolysis in Human Plasma Induced by Dextran Sulfate

The analysis of normal human plasma by fibrin autography revealed four species of plasminogen activator (PA) activity related to tissue-type PA, factor XII, prekallikrein and urokinase-type PA (u-PA). The u-PA activity increased significantly by incubating plasma with dextran sulfate. This increase was coincident with both the cleavage of factor XII and the complex formation of activated factor XII with its plasma inhibitors, which were determined by immunoblotting procedure. The dextran sulfate-dependent activation of u-PA required both factor XII and prekallikrein, but did not require either plasminogen or factor XI. High molecular weight kininogen was required only at a low concentration of dextran sulfate. Thus the results indicate that the factor XII and prekallikrein-mediated activation of single chain u-PA (scu-PA) operates as a major pathway of scu-PA activation in whole plasma in contact with dextran sulfate.

Charge selectivity in kidney ultrafiltration is associated with glomerular uptake of transport probes

The isolated perfused kidney exhibits substantial charge selectivity, as in vivo, in relation to fractional clearance of [3H]dextran sulfate and [3H]dextran. When cycloheximide is present in perfusate, fractional clearance of dextran sulfate is increased and proteinuria becomes significant, but glomerular filtration rate remains essentially unchanged compared with control. The possible role of cells in affecting transglomerular transport was demonstrated when isolated glomeruli from control perfused kidneys showed a very significant resident concentration of [3H]dextran sulfate and [3H]albumin, whereas there was no corresponding accumulation of [3H]dextran or [3H]inulin. Glomerular concentration of dextran sulfate and albumin was significantly reduced by cycloheximide. Kinetics of uptake and release of glomerular dextran sulfate indicated that it had a half-life of glomerular residence of approximately 2-3 min and that this half-life was considerably extended in the presence of cycloheximide. The half-life for glomerular residence of albumin was in the range of 30-40 min. The conclusion from this work is that glomerular charge selectivity for dextran sulfate could be quantitatively rationalized on the basis of transient uptake and release by glomerular cells.

Negative Supercoiling of DNA by Eukaryotic DNA Topoisomerase II and Dextran Sulfate<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

In the presence of a molar excess of eukaryotic DNA topoisomerase II and an appropriate concentration of dextran sulfate, relaxed closed circular DNA is converted to a negatively supercoiled form. The reaction is dependent on ATP. Neither adenosine 5'-[beta,gamma-imido]-triphosphate nor adenosine 5'-[gamma-thio]triphosphate can substitute for ATP. The negative supercoils formed are relaxed by subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of a nicked circular DNA in the presence of DNA topoisomerase II and dextran sulfate but in the absence of ATP causes a small decrease in the linking number. These results suggest that when an appropriate concentration of dextran sulfate is present, the binding of a molar excess of eukaryotic DNA topoisomerase II constrains a small number of negative supercoils in DNA, which in turn generate unconstrained negative supercoils at the expense of ATP.

Improved Storage Methods for Epikeratoplasty

A major variable in calculating the necessary curvatures and dimensions of the refractive lenticules for epikeratoplasty is the degree of swelling of the donor cornea during storage prior to lathing. The purpose of this study was to determine whether it is possible to restore donor corneas to their in vivo thickness using osmotic agents rather than physical means. The osmotic agents chosen were dextran and chondroitin sulfate. Donor porcine corneas stored in McCarey-Kaufman (MK) medium for seven days increased in thickness by almost 50%. Increasing concentrations of dextran and chondroitin sulfate, and increasing concentrations of dextran with 1.35% chondroitin sulfate added, were examined for their ability to reduce this thickness to normal levels. It was found that 15.00% dextran alone, and 10.00% dextran with 1.35% chondroitin sulfate, reduced corneal thickness to that of fresh tissue. Light and electron microscopy showed that the osmotic agents restored stromal ultrastructure to that of fresh tissue. The use of osmotic agents to reduce corneal thickness prior to lathing reduces physical deformation of the donor tissue associated with the use of a corneal press and may enhance the predictability of epikeratoplasty.

Anti-human immunodeficiency virus effects of dextran sulfate are strain dependent and synergistic or antagonistic when dextran sulfate is given in combination with dideoxynucleosides

The effects of three molecular weight ranges of dextran sulfate on five different human immunodeficiency virus (HIV) isolates (from patients with acquired immunodeficiency syndrome), alone and in combination with dideoxynucleosides, were investigated in vitro. The higher the molecular weight range of dextran sulfate, the more potent the activity as assessed by a quantitative syncytium formation assay. Although all five HIV isolates had similar susceptibilities to the inhibitory effects of dideoxynucleosides, the two clinical isolates of HIV (HIV type 1 [HIV-1] TM and SP) exhibited a pattern of reduced susceptibility to dextran sulfate when compared with the two cloned isolates (HIV-1 WMF and HIV-2 ROD) and a prototype laboratory strain (HIV-1 IIIB). In combination with dideoxynucleosides, the high-molecular-weight range of dextran sulfate (500,000) resulted in an antagonistic response directed against the two clinical isolates of HIV (HIV-1 TM and SP) when the antiviral concentrations of dextran sulfate were in the ineffective range. Additive or synergistic effects were seen with the other three HIV isolates and all five HIV isolates when the low-molecular-weight range of dextran sulfate (8,000) was used. The results of these studies raise issues on the impact of drug-resistant strains on disease progression and the use of dextran sulfate in combination with nucleoside analogs for the clinical management of HIV disease.

Interaction of dextran sulfate with phospholipid surfaces and liposome aggregation and fusion

The binding of dextran sulfate to phospholipid liposomes was investigated by microelectrophoresis experiments. The polyanion binds to neutral phospholipd liposomes (DMPC and PE) only in the presence of Ca 2+. If positively charged stearylamine is incorporated in the vesicles dextran sulfate is bound without Ca 2+. Negatively charged phospholipids as PS do not bind dextran sulfate, even in the presence of millimolar concentrations of Ca 2+. The adsorption of dextran sulfate results in an aggregation of vesicles due to a bridging mechanism. In all cases the aggregation is followed by a disaggregation toward higher dextran sulfate concentrations. The disaggregation process starts at polymer concentration smaller than the concentration of the onset of saturation of the adsorption. By use of the prove dilution method a fusion of small DMPC and DMPC/PE vesicles in the presence of Ca 2+ and dextran sulfate was found

Sodium Pentosan Polysulfate (PPS), an Anti-HIV Agent Also Exhibits Synergism with AZT, Lymphoproliferative Activity, and Virus Enhancement

Sodium pentosan polysulfate (PPS), a negatively charged polymer of beta-D-xylopyranose units, was evaluated for its anti-HIV effects in normal human peripheral mononuclear cells (PMNC) and its possible synergism with AZT. In the presence of 25 nM AZT, 2.0 micrograms/ml of PPS reduced HIV-1 replication 110-fold, compared with a 3.9- and 7-fold decrease in the presence of either drug individually. Surprisingly, at low (below 1 microgram/ml) concentrations of either PPS or dextran sulfate, an enhancement of virus production was observed. PPS was nontoxic, had a proliferative effect on uninfected and a protective effect on infected PMNC. Virus enhancement at low concentrations of PPS appeared to be linked to its lymphoproliferative effect. These findings suggest that the use of PPS and others such agents as monotherapy for AIDS might have deleterious effects. However, due to its marked synergism with AZT and its lymphoproliferative activities, PPS might prove to be a useful agent in therapeutic trials of AIDS if used in combination with less than the usual dosage of AZT.

Use of a dual-precipitation procedure for measuring high-density lipoprotein 3 (HDL3) in normolipidemic serum.

We compared results by a dual-precipitation method (J Lipid Res 1982;23:1206-23) for measuring high-density lipoprotein 3 (HDL3) cholesterol with those by ultracentrifugation at d 1.125, using 56 fresh and 105 frozen-stored serum samples. For both methods, HDL2-cholesterol was calculated as the difference between total HDL-cholesterol and HDL3-cholesterol. In general, for pooled serum samples, agreement was closest with ultracentrifugation when we used a dextran sulfate concentration of 5.0 mg/L to precipitate the HDL2-rich fraction, although the optimal concentration varied from 3.0 to 6.8 mg/L for different pools. For individual samples, the values for HDL3 by dual precipitation averaged 12.8% lower than by ultracentrifugation. The coefficients of correlation between the two methods were HDL3, r = 0.70; and HDL2, r = 0.92. The dual-precipitation method reflected the expected sex-related differences in HDL2-cholesterol concentration and inverse relationship with triglyceride concentration.

Determination of HDL2 cholesterol by precipitation with dextran sulfate and magnesium chloride: Establishing optimal conditions for rat plasma

Optimal conditions for analyzing HDL2 cholesterol in small amounts of rat plasma have been studied using different concentrations of dextran sulfate and MgCl2 to precipitate lipoproteins containing apolipoprotein B and/or apo E. When the MgCl2 level was 91 mM, the supernate cholesterol was rather constant at a level of about 50-60% of the total plasma cholesterol concentration. Immunochemical determination of the apo A-I content indicated that no major losses of the HDL2 fraction took place under these conditions. The recovery of about 96% of HDL2 lipoproteins after the precipitation of rat plasma and the almost complete absence of lipoproteins belonging to the VLDL, LDL and HDL1 fractions was demonstrated by agarose gel electrophoresis. Thus, the method should be suitable for screening the HDL2 cholesterol content in small volumes of rat plasma.

Regulation of 12-hydroxyeicosatetraenoic acid synthesis by acetyl-LDL in mouse peritoneal macrophages

The mechanism for the regulation of 12-hydroxyeicosatetraenoic acid (12-HETE) production by cholesterol-rich macrophages was investigated. β-VLDL and acetyl-LDL, lipoproteins which result in cholesterol accumulation in macrophages, stimulated 12-HETE secretion. Lipoproteins which do not induce cholesterol accumulation, such as low- and high-density lipoproteins, did not. Cell-free homogenates from cholesterol-rich macrophages had significantly more 12-lipoxygenase activity than homogenates from unmodified cells. Preincubating homogenates prepared from unmodified macrophages with acetyl-LDL, LDL or multilamellar liposomes containing total lipids from acetyl-LDL but not apoproteins significantly increased 12-lipoxygenase activity. This stimulatory effect was caused by the phospholipid molety of the lipoprotein. 12-HETE synthesis was not increased in macrophages enriched 6-fold in unesterified cholesterol. Acetyl-LDL stimulated 12-HETE synthesis in macrophages in which cholesteryl ester accumulation was prevented by inhibiting acylcoenzyme A: cholesterol acyltransferase activity. When binding of acetyl-LDL to its receptor was decreased by increasing concentrations of dextran sulfate, or when lysosomal metabolism of the lipoprotein was prevented by chloroquine, 12-HETE production significantly decreased. Moreover, the combination of inhibiting acetyl-LDL binding and degradation completely blocked the stimulation of 12-HETE synthesis by acetyl-LDL. The data indicate that acetyl-LDL must enter the macrophage and be partially degraded to regulate 12-HETE synthesis. The regulation is independent of cholesterol accumulation but is related to the entering lipoprotein phospholipid.

Interference of blood clotting factors in the determination of HDL 2- and HDL 3-cholesterol by the dual precipitation method

Previously we found that in the dual precipitation method for determination of HDL 2- and HDL 3-cholesterol the optimal concentration of dextran sulfate was 0.87 g/l instead of 1.3 or 1.5 g/l. In the present study, we performed the dual precipitation method both in serum and in EDTA-plasma samples in order to investigate whether the method is sensitive for plasma-serum differences. It was found, that in the analysis of EDTA-plasma samples a twice higher final concentration of dextran sulfate was needed. Addition of the dextran sulfate resulted in the appearance of fibrin structures in the HDL-fraction which may have diminished the effective dextran sulfate concentration in the precipitation reaction. Although the use of plasma is advised in the analysis of lipoproteins, the use of EDTA-plasma in precipitation methods for determination of HDL-cholesterol and its subfractions cannot be recommended.

Evaluation of the dual-precipitation method for determination of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 in serum.

We compared the dual-precipitation method for measurement of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 (Gidez et al., J Lipid Res 1982;23:1206-33) with density-gradient ultracentrifugation in a swinging-bucket rotor (Demacker et al., Clin Chem 1983;29:656-63). The concentration of dextran sulfate 15,000 (DS) needed for optimal accuracy of the HDL2-chol and HDL3-chol values was established empirically. At a DS concentration of 0.87 g/L, the values for HDL2-chol as well as for HDL3-chol in 88 sera did not differ significantly from those obtained by ultracentrifugation. The precision of the method was satisfactory and was related to the concentration. Nevertheless, the dual-precipitation method lacks specificity inasmuch as it produces no fractions that contain only one HDL subfraction. HDL2 and HDL3 each contained an equivalent amount of cholesterol from the other. At increasing DS concentrations, some radiolabeled HDL3 appeared to have precipitated prior to complete precipitation of HDL2. This lack of specificity can be tolerated in large-scale epidemiological studies for screening, but not in small-scale intervention studies or in assay of clinical samples, where better accuracy is needed and ultracentrifugation is preferred.