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G Torri

Publications and source records attributed to G Torri.

At least 163 records · Page 9Linked to original sources
PubMed1990

Biosynthesis of heparin. Availability of glucosaminyl 3-O-sulfation sites.

Heparin preparations isolated from pig intestinal mucosa and from bovine lung were fractionated with regard to affinity for antithrombin. The resulting fractions, with high (HA) or low (LA) affinity for the proteinase inhibitor, were analyzed by 13C NMR or by identification of di- and tetrasaccharides obtained through deaminative cleavage with nitrous acid. Structural differences between corresponding HA and LA fractions were essentially restricted to minor constituents, in particular 3-O-sulfated glucosamine units that occurred (1 or 2 residues/chain) in all HA preparations but were scarce or absent in LA heparin. The HA fractions also consistently showed higher contents of nonsulfated iduronic acid and, to a lesser extent, N-acetylated glucosamine units than the LA fractions. The two tetrasaccharide sequences, -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3- and -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3(6-OSO3)- , recently implicated as part of the acceptor site for glucosaminyl 3-O-sulfate groups (Kusche, M., Bäckström, G., Riesenfeld, J., Petitou, M., Choay, J., and Lindahl, U. (1988) J. Biol. Chem. 263, 15474-15484), were identified in mucosal LA heparin; it was calculated that the preparation contained approximately one potential acceptor site/polysaccharide chain. Yet this material did not yield any labeled HA components on incubation with adenosine 3'-phosphate 5'-phospho-[35S]sulfate in the presence of glucosaminyl 3-O-sulfotransferase, solubilized from a mouse mastocytoma microsomal fraction. The failure to incorporate any 3-O-sulfate groups could conceivably be explained by the occurrence of a D-glucuronic rather than L-iduronic acid unit linked at the reducing ends of the above tetrasaccharide sequences. Alternatively, 3-O-sulfation may be restricted by other, as yet unidentified, inhibitory structural elements that are preferentially expressed in polysaccharide sequences selected for the generation of LA heparin.

Animals↗
PubMed1990

Conformer populations of L-iduronic acid residues in glycosaminoglycan sequences.

The 1H-n.m.r. 3J values for the L-iduronic acid (IdoA) residues for solutions in D2O of natural and synthetic oligosaccharides that represent the biologically important sequences of dermatan sulfate, heparan sulfate, and heparin have been rationalized by force-field calculations. The relative proportions of the low-energy conformers 1C4, 2S0, and 4C1 vary widely as a function of sequence and of pattern of sulfation. When IdoA or IdoA-2-sulfate units are present inside saccharide sequences, only 1C4 and 2S0 conformations contribute significantly to the equilibrium. This equilibrium is displaced towards the 2S0 form when IdoA-2-sulfate is preceded by a 3-O-sulfated amino sugar residue, and towards the 1C4 form when it is a non-reducing terminal. For terminal non-sulfated IdoA, the 4C1 form also contributes to the equilibrium. N.O.e. data confirm these conclusions. Possible biological implications of the conformational flexibility and the counter-ion induced changes in conformer populations are discussed.

Carbohydrate Conformation↗
PubMed1989

Respiratory mechanics in anesthetized paralyzed humans: effects of flow, volume, and time.

The effects of inspiratory flow rate and inflation volume on the resistive properties of the total respiratory system were investigated in 16 anesthetized paralyzed humans by using the technique of rapid airway occlusion during constant flow inflation. This allowed measurement of the intrinsic resistance (Rmin,rs) and of the effective additional resistance (delta Rrs) as the result of viscoelastic pressure dissipations within the pulmonary and chest wall tissues. We observed that 1) at fixed inflation volume, Rmin,rs increased linearly with increasing flow although delta Rrs decreased according to an exponential function; 2) at fixed inflation flow, Rmin,rs decreased with increasing inflation volume although there was a concomitant increase in delta Rrs. This behavior could be explained in terms of a spring-and-dashpot model incorporating 1) the standard resistance and elastance and 2) a spring-and-dashpot in parallel with standard elastance, reflecting the stress adaptation units within the thoracic tissues.

Adolescent↗
PubMed1987

"Supersulfated" heparin fragments, a new type of low-molecular weight heparin. Physico-chemical and pharmacological properties.

A new type of low-molecular-weight heparin (ss-LMW-H) was prepared (by controlled depolymerization and concurrent sulfation of heparin with a mixture of sulfuric and chlorosulfonic acid), to test the influence of extra-sulfate groups on biological properties of heparin fragments. The fragments had an average molecular weight ranging from 5000 to 10,000, a sulfate-to-carboxyl molar ratio of 2.8-3.1, and electrophoretic mobilities and NMR spectra distinctly different from those of the parent heparins. Depolymerization with oversulfation reduced the anticoagulant activity of heparin (ex vivo, in rats) much more than depolymerization alone, to about 10% of the original APTT and 25-30% of the original a.Xa units. By contrast, the antithrombotic activity (venous stasis model, in rats) was still comparable to that of heparin, and bleeding times were not significantly increased. The lipasemic (lipoprotein-lipase-releasing) activity of ss-LMW-H fragments was more than twice that of heparin. Results are discussed in terms of contribution of charge-density effects to different activities and to different mechanisms for the same activity of heparin.

Animals↗
PubMed1986

Synthesis of heparin fragments. A chemical synthesis of the pentasaccharide O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1-4 )-O-(beta-D-glucopyranosyluronic acid)-(1-4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-glu copyranosyl)-(1-4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-4)-2-deoxy-2-sulfamido-6-O-sulfo-D-glucopyranose decasodium salt, a heparin fragment having high affinity for antithrombin III.

Known allyl 4,6-O-benzylidene-alpha-D-glucopyranoside was first converted into methyl (prop-1-enyl 2,3-di-O-benzyl-4-O-chloroacetyl-alpha-D-glucopyranosid)-uronate. Acid hydrolysis, followed by treatment with (bromomethylene)dimethyl-ammonium bromide, gave methyl (2,3-di-O-benzyl-4-O-chloroacetyl-alpha-D-glucopyranosyl bromide)uronate. Condensation of this bromide with 3-O-acetyl-1,6-anhydro-2-azido-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-bet a-D-glucopyranose. Acetolysis, followed by treatment with titanium tetrabromide, then gave 3,6-di-O-acetyl-2-azido-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-alp ha-D-glucopyranosyl bromide. Condensation of this bromide with benzyl 6-O-acetyl-3-O-benzyl-2-benzyloxy- carbonylamino-2-deoxy-4-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L- idopyranosyluronate)-alpha-D-glucopyranoside provided benzyl O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-(1- ---4)-O-(3,6-di-O-acetyl- -2-azido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-ac etyl-3-O- acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-gluc opyranoside. O-Dechloroacetylation followed by condensation with 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide provided benzyl O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-beta-D-glucopyranosyl)- (1----4)-O-(methyl 2,3-di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)- O-(3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(m ethyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-ac etyl-3-O- benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-glucopyranoside in 70% yield. O-Deacetylation followed by re-esterification, O-sulfation, saponification, catalytic hydrogenolysis, and N-sulfation gave the decasodium salt of O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)-(1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gl ucopyranosyl)-(1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic+ ++ acid)-(1----4)-2-deoxy-2-sulfamido-6-O-sulfo-D-glucopyranose. This synthetic pentasaccharide binds to antithrombin III with an association constant similar to that of high-affinity heparin and elicits a potent anti-factor Xa activity in plasma.

Antithrombin III↗
PubMed1986

Retention of antilipemic activity by periodate-oxidized non-anticoagulant heparins.

Heparin preparations with different anticoagulant and antilipemic (fat-clearing) activities were oxidized with periodate under conditions of cleavage of all the C(2)-C(3) bonds of non-sulfated uronic acid residues, while preserving the original molecular weight of the polysaccharide. Periodate-oxidised heparins (oxyheparins, O-HEP) and the corresponding borohydride-reduced products (reduced oxyheparins, RO-HEP) were compared with the original heparins for their content in trisulfated disaccharide sequences (as determined by 13C-nuclear magnetic resonance) and in active sites for antithrombin-III (as determined indirectly by affinity chromatography), and for their anticoagulant and antilipemic (lipoprotein lipase-releasing) activities. The drop of anticoagulant activity induced by periodate oxidation was paralleled by a substantial decrease of affinity for antithrombin, and is thought to arise from glycol splitting at the level of the D-glucuronic acid residue that is part of the active site for antithrombin. The trisulfated disaccharide sequences and the associated antilipemic activities were substantially unaffected by periodate oxidation. The residual anticoagulant activity of periodate-oxidized heparins obtained from preparations - such as those from beef lung - rich in trisulfated disaccharide sequences is discussed in terms of the influence of charge density on heparin-protease interactions not mediated by antithrombin.

Animals↗
PubMed1985

Antithrombotic and bleeding effects of a low molecular weight heparin fraction.

Low molecular weight (LMW) heparin prevents venous thrombosis by potentiating the inhibition of coagulation factor Xa. Heparin, however, has other biological properties whose role in the prevention of thrombosis is still unknown. The aim of our study was to compare the antithrombotic activity of a LMW heparin and its parent molecule in an attempt to understand better the mechanism and structural requirements for heparin's antithrombotic effect. We studied a preparation of an unfractionated pig mucosal heparin pure by any accepted criteria (electrophoresis in various systems, conductimetric titration and NMR spectra) and a LMW heparin fraction obtained from the former by fractional precipitation with ethanol. Both heparins completely prevented thrombus formation in an experimental model of stasis-induced venous thrombosis in rats. When administered intravenously to rats, the unfractionated heparin had an ex vivo anti-Xa/APTT ratio of 1.67, versus 6.60 of the LMW heparin fraction. Unexpectedly, both heparins induced a significant prolongation of tail bleeding time, performed by two different techniques, the "transection" (mostly exploring blood clotting) and the "template" (exploring the platelet/vessel wall interactions). This study suggests that, beside anticoagulation, other effects may play a role in both the antithrombotic and haemorrhagic effects of some heparins and LMW heparin fractions.

Animals↗
PubMed1985

Mono- and bidimensional 500 MHz 1H-NMR spectra of a synthetic pentasaccharide corresponding to the binding sequence of heparin to antithrombin-III: evidence for conformational peculiarity of the sulfated iduronate residue.

1H-NMR spectra of the synthetic pentasaccharide (N-sulfate-6-0-sulfate-alpha-D-glucosamine) 1----4 (beta-D-glucuronic acid) 1----4 (N-sulfate-3,6-di-0-sulfate-alpha-D-glucosamine) 1----4 (2-0-sulfate-alpha-L-iduronic acid) 1----4 (N-sulfate-6-0-sulfate-alpha-D-glucosamine), corresponding to the active site of heparin for antithrombin (AT-III), have been resolved at 500 MHz and assigned by mono- and bidimensional techniques. Vicinal proton coupling constants of the D-glucosamine residues are similar to those in the regular sequences of heparin, indicating that the 4C1 conformation of the ring, and preference for the g,g conformation of the sulfated hydroxymethyl groups of these residues are neither affected by the unique 3-0-sulfo group nor by sequence effects. By contrast, an unusually large coupling between H-2 and H-3 of the sulfated L-iduronic acid residue suggests a greater departure from the 1C4 conformation of this residue. when present in the binding sequence to AT-III than in the regular sequences. Such a departure, leading to different orientation and spacing of essential sulfate groups, may have implications for high-affinity binding to AT-III.

Antithrombin III↗
PubMed1985

Specific antibodies to diphtheria toxin and type 6A pneumococcal capsular polysaccharide induced by a model of semi-synthetic glycoconjugate antigen.

A molecular model of a carbohydrate-protein conjugate is described, involving the non-toxic mutant protein CRM197, serologically related to the diphtheria toxin, covalently bound to a characterized oligosaccharide derived from the molecular structure of type 6A pneumococcal capsular polysaccharide. Physicochemical and immunochemical characteristics of this oligosaccharide-protein conjugate were consistent with a molecule showing a molar carbohydrate/protein ratio of 8, an av. mol. wt of 75,000, and retention of complete immunochemical identity when tested towards the homologous antisera. The immunological characteristics obtained after immunization of 2 animal models showed a high immunogenicity of the glycoconjugate specifically directed towards diphtheria toxin and the type 6A pneumococcal capsular polysaccharide.

Animals↗