Potentiation of the action of fibroblast growth factor by heparin and related molecules.
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3T3 Cells↗
Biomedical subjects
B Mulloy
Publications and source records attributed to B Mulloy.
Structural diversity among sulfated alpha-L-galactans from ascidians (tunicates). Studies on the species Ciona intestinalis and Herdmania monus.
Sulfated polysaccharides occurring in the tunic of different species of ascidians differ markedly in electrophoretic pattern and chemical composition. A purified sulfated alpha-L-galactan from Herdmania monus was studied using methylation analysis and NMR spectroscopy; it is composed mainly of 3-sulfated 4-linked alpha-L-galactopyranoyl units. This is the first description of a homo-polymer of sulfated alpha-L-galactose. In contrast, the sulfated L-galactan from Ciona intestinalis shows marked structural heterogeneity and a low sulfate content. These data indicate unusual structural diversity among sulfated L-galactans from different species of ascidians.
Animals↗
Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues.
The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.
Animals↗
Characterization of inositol 1,4,5-trisphosphate- and inositol 1,3,4,5-tetrakisphosphate-binding sites in rat cerebellum.
1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude 'P2' cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.
Animals↗
Structure of a unique sulfated alpha-L-galactofucan from the tunicate Clavelina.
A purified sulfated alpha-L-galactofucan from Clavelina sp. has been studied before and after desulfation, using periodate oxidation, methylation analysis, and n.m.r. spectroscopy, and shown to be composed mainly of 3-sulfated 4-linked alpha-L-galactopyranosyl residues. There was also a small proportion of 3-sulfated 4-linked alpha-L-fucose residues.
Animals↗
Structural studies of a sulfated L-galactan from Styela plicata (Tunicate): analysis of the Smith-degraded polysaccharide.
The tunic of the ascidian Styela plicata is rich in a high molecular weight sulfated-L-galactan called the F-1 fraction. This polysaccharide is of complex structure and is highly branched. In this study we undertook detailed structural analysis of the F-1 fraction that was submitted to the Smith degradation procedure which, for this polysaccharide, results mainly in the elimination of the branches. By methylation, and one- and two-dimensional n.m.r. analysis of the Smith-degraded and desulfated Smith-degraded F-1 fraction, we determined the main structure of this polymer. It is composed of a core of alpha-L-galactopyranose units, sulfated at C-3 and glycosidically linked through position 1----4. The nonsulfated, nonreducing end units are branched at C-2 of the sulfated L-galactoses.
Alcohols↗
Antimicrobial effect of human milk on Bordetella pertussis.
It has been demonstrated that human milk, unlike bovine milk, can reduce the viability of Bordetella pertussis. This antibacterial activity was not due to the presence of antibiotics or antibodies in the human milk. Reducing the level of available iron or increasing the concentration of lysozyme in bovine milk did not induce anti-B. pertussis activity. Analysis of total fatty acids revealed that human milk contained significantly more linoleic acid than bovine milk. However, the addition of linoleic acid to bovine milk did not inhibit the growth of B. pertussis.
Animals↗
NOEMOL: integrated molecular graphics and the simulation of Nuclear Overhauser effects in NMR spectroscopy.
Nuclear Overhauser effects (NOEs) are a widely used method of determining the spatial proximity of spins in Nuclear Magnetic Resonance (NMR) spectroscopy. This paper describes a C program developed for the Sun-3 workstation family that allows the computation of multispin NOE effects for a given molecular structure and given NMR parameters (i.e., resonance frequency and correlation time for molecular reorientation). The integration of these facilities with simple molecular graphics display routines allows modifications to the molecular conformation (such as bond rotations) to be performed, and the effect of these modifications on the NOE effects can then be rapidly calculated and easily visualized. Using the Sun windowing system, the NOE effects can be calculated for two (or more) candidate structures and compared to experimental NMR results. The overall molecular reorientation can be modeled by either isotropic or symmetric top diffusion models, and the internal motions of methyl groups are modeled using an algorithm reported by Tropp.
Carbohydrate Sequence↗
The anticoagulant activity of heparin: measurement and relationship to chemical structure.
For many years the anticoagulant activity of heparin has been estimated by coagulation assays, in which the prolongation of clotting times by heparin is measured under various conditions. More recently, assays have been developed which measure the inhibitory action of heparin on isolated coagulation enzymes, notably Factor Xa and thrombin, using specific amidolytic peptide substrates. The anticoagulant activity of heparin arises primarily from its ability to bind to antithrombin III (AT III), altering the conformation and enhancing the activity of this major protease inhibitor. Passage of heparin through an immobilised AT III column yields two fractions: a high affinity fraction with 300-350 iu mg-1 anticoagulant activity, comprising one-third of the total, and a low affinity fraction with an activity of less than 10 iu mg-1, comprising the remaining two-thirds. Studies in several laboratories have demonstrated that a specific pentasaccharide sequence is required for AT III binding. The authors have shown that the presence or absence of this sequence can be detected by high-field proton NMR, thus providing a semi-quantitative method for a functionally important group. A second major influence on anticoagulant activity is molecular weight distribution. Studies in the authors' laboratory on a series of fractions of 5000-35,000 showed that whereas anticoagulant activity in APTT clotting assays decreased with decreasing molecular weight (Mr), activity in anti-Xa assays was maintained or increased in the low Mr fractions. However, in vivo studies showed that high affinity fragments with anti-Xa activity only were poor antithrombotic agents. It appears that the presence of the AT III binding site alone is not sufficient for full antithrombotic activity; an extra length of polysaccharide chain of at least 15 residues is required. Molecular weight distribution is readily assessed by HPLC, although the lack of suitable reference materials hampers assignment of absolute molecular weights. Important determinants of anticoagulant activity can now be assessed by physicochemical techniques but, at present, these techniques are not precise enough to replace anticoagulant assays as predictors of in vivo behaviour.
Heparin↗
Long-range carbon-proton coupling constants: application to conformational studies of oligosaccharides.
3JC,H values have been measured using selective 2D heteronuclear J-resolved n.m.r. spectroscopy for the COCH fragment in various carbohydrates. Measurements on model compounds have been used to characterise a Karplus-type relationship between 3JCOCH and dihedral angles in sugar. The 3JC,H values have also been measured for C-2'-O-2'-C-1-H-1 of sucrose and the sucrose residues in raffinose, stachyose, and melezitose. These values are similar to each other for solutions in D2O and (CD3)2SO and are little affected by the change in solvent, but differ from those predicted from the crystal conformations. The method has been used to correct some assignments in the published 13C-n.m.r. spectrum of melezitose.
Carbohydrate Conformation↗
Assignment of the 1H-n.m.r. spectra of heparin and heparan sulphate.
Resonances from the main repeating unit of heparan, ----4)-beta-D-GlcA-(1----4)-alpha-D-GlcNAc-(1----, have been assigned by using a sample of the capsular polysaccharide of E. coli K5. Comparison of the spectra of heparan sulphate samples before and after O- and/or N-desulphation, with re-N-acetylation or re-N-sulphation, allowed assignment of some of the H-1 doublets in terms of sequence effects. Chemical shifts for H-1 of unsulphated uronic acid residues are influenced by 6-sulphation of the nearest neighbour GlcN on the reducing side; those of GlcN residues vary according to whether they have IdoA or GlcA as the nearest neighbour on the reducing side. The H-1 doublets due to residues in the binding sequence for antithrombin have been assigned by comparison of the spectra of heparins having high and low affinities for immobilised antithrombin.
Animals↗
Correlation between structure, fat-clearing and anticoagulant properties of heparins and heparan sulphates.
Heparins and heparan sulphates from different organs, of high purity by accepted criteria, were characterized by chemical and physical (including electrophoretic and 13C-NMR spectroscopic) methods. The fat-clearing activity of these preparations was shown to be correlated with their content of the trisulphated disaccharide units I2S-ANS,6S (L-iduronic acid 2-O-sulphate-N-sulphated D-glucosamino 6-O sulphate). Species with low anti-lipemic activity contained significant proportions (greater than or equal to 30%) of nonsulphated uronic acids (especially D-glucuronic acid), and of D-glucosamino residues undersulphated at C-6, these latter residues being partially N-acetylated. Heparins from beef lung and sheep mucosa, predominantly consisting of trisulphated disaccharide units, displayed consistently higher antilipemic activity than the more heterogeneous pig mucosal heparins. The anticoagulant activity (as determined by the U.S.P., APTT and anti-Xa tests) was not a simple function of the measured physicochemical parameters. Trends were confirmed for pig mucosal heparins being more anticoagulant than beef lung preparations, and low molecular weight (usually undersulphated) species being more active in the anti-Xa test than in the U.S.P. and APTT tests.
Amino Acids↗
Gel permeation chromatography of heparins.
Gel permeation chromatography of heparins using water as solvent has recently been used to obtain fractions for biological investigation. We find that heparin fractions obtained in this way are not, necessarily, differentiated simply on a molecular size basis as has been alleged. Correlations between biological activity and molecular size obtained using such fractions must therefore be treated with caution.
Animals↗
The molecular-weight range of mucosal-heparin preparations.
A recently reported method describes the determination of the molecular-weight range of heparins by using an electrofocusing procedure to isolate individual molecular species. Commercially available heparins were fractionated on a column of polyacrylamide-agarose gel to give fractions whose molecular weights were estimated by viscometry. Fractions with mutually exclusive molecllar-weight ranges gave an appreciable number of common bands when subjected to the electrofocusing procedure; therefore, each of these bands cannot be formed from a single molecular species of heparin. Other mucopolysaccharides also gave band sequences indistinguishable from those of heparin; they differed in position and intensity with different ampholyte batches, and probably arose from individual molecular species of the ampholyte rather than the mucopolysaccharide. The molecular-weight range of the heparin was observed to be broader than that usually reported.
Chromatography, Gel↗
Simple metachromatic assay methods for heparin and protamine.
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Chondroitin↗
Anti-factor Xa activity of heparan sulphate.
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Animals↗
Heparin inhibition of human vascular smooth muscle cell hyperplasia.
BACKGROUND: Vascular smooth muscle cell (VSMC) growth is responsible for intimal hyperplasia, a major cause of failure after vascular surgery and angioplasty. Heparin is the first described inhibitor of VSMC growth, but has not proved effective in the prevention of human intimal hyperplasia. Heparin is a heterogeneous substance, which may contain a mixture of components which differ in antiproliferative activity. Isolation of an active component may favourably influence its therapeutic profile. METHODS AND RESULTS: Growth of human VSMC cultured from operative specimens, assessed by cell counting and labelled thymidine incorporation, was used as a model of VSMC proliferation in intimal hyperplasia. Unfractionated (UFH) and low molecular weight (LMWH) heparins inhibit cell growth and thymidine uptake by human VSMCs in response to 15% foetal calf serum. UFHs are more active than LMWHs and this difference increases with increasing heparin dose. To confirm this effect, size-based fractions of heparin were prepared by gel permeation chromatography, and characterised by high performance liquid chromatography. High molecular weight fractions (MW > 21000) have higher activity than fractions of medium (MW 12000-21000) or low molecular weight (MW < 12000). These differences become more pronounced at higher dose, and are statistically significant at 100 micrograms/ml (Mann-Whitney, p < 0.05). CONCLUSIONS: The antiproliferative activity of heparin appears to be maximal in its high molecular weight component.
Cell Division↗