Biomedical subjects
Publications and source records attributed to A Bisio.
From the exudate of Salvia wagneriana Polak, beside the known ursolic acid, two pentaoxygenated triterpenoids were isolated. Their structures were determined as 3-oxo-11alpha,19beta,20,22beta-tetrahydroxy-lupane (1) and 3beta,11alpha,19beta,20,22beta-pentahydroxy-lupane (2) using a combination of one- and two-dimensional NMR techniques.
In the framework of a project aimed at generating heparin-like sulfation patterns and biological activities in biotechnological glycosaminoglycans, different approaches have been considered for simulating the alpha(1-->4)-linked 2-O-sulfated L-iduronic acid (IdoA2SO(3))-->N,6-O-sulfated D-glucosamine (GlcNSO(3)6SO(3)) disaccharide sequences prevalent in mammalian heparins. Since the direct approach of sulfating totally O-desulfated heparins, taken as model compounds for C-5-epimerized sulfaminoheparosan (N-deacetylated, N-sulfated Escherichia coli K5 polysaccharide), preferentially afforded heparins O-sulfated at C-3 instead than at C-2 of the iduronate residues, leading to products with low anticoagulant activities, the problem of re-generating a substantial proportion of the original IdoA2SO(3) residues was circumvented by performing controlled solvolytic desulfation (with 9:1 v/v DMSO-MeOH) of extensively sulfated heparins. The order of desulfation of major residues of heparin GlcN and IdoA and of the minor one D-glucuronic acid was: GlcNSO(3)>GlcN6SO(3)>IdoA3SO(3) congruent with GlcA2SO(3) congruent with GlcN3SO(3)>IdoA2SO(3) congruent with GlcA3SO(3). Starting from a 'supersulfated' low-molecular weight heparin, we obtained products with up to 40% of iduronate residues O-sulfated exclusively at C-2 and up to 40% of their glucosamine residues O-sulfated at both C-6 and C-3. Upon re-N-sulfation, these products displayed an in vitro antithrombotic activity (expressed as anti-factor Xa units) comparable with those of current low-molecular weight heparins.
The interaction, in aqueous solution, of the synthetic pentasaccharide AGA*IA(M) (GlcN,6-SO(3)alpha 1-4GlcA beta 1-4GlcN,3,6-SO(3)alpha 1-4IdoA,2-SO(3)alpha 1-4GlcN,6-SO(3)alpha OMe; where GlcN,6-SO(3) is 2-deoxy-2-sulphamino-alpha-D-glucopyranosyl 6-sulphate, IdoA is l-iduronic acid and IdoA2-SO(3) is L-iduronic acid 2-sulphate), which exactly reproduces the structure of the specific binding sequence of heparin and heparan sulphate for antithrombin III, has been studied by NMR. In the presence of antithrombin there were marked changes in the chemical shifts and nuclear Overhauser effects (NOEs), compared with the free state. On the basis of the optimized geometry of the pentasaccharide the transferred NOEs were interpreted with full relaxation and conformational exchange matrix analysis. An analysis of the three-dimensional structures of the pentasaccharide in the free state, and in the complex, revealed the binding to be accompanied by dihedral angle variation at the A-G and I-A(M) (where G, I, A and A(M) are beta-d-glucuronic acid, 2-O-sulphated alpha-L-iduronic acid, N,6-O-sulphated alpha-D-glucosamine and the alpha-methyl-glycoside of A respectively) glycosidic linkages. Evidence is also provided that the protein drives the conformation of the 2-O-sulphated iduronic acid residue towards the skewed (2)S(0) form.
Several preparations of low molecular weight heparins (LMWHs) obtained by physical depolymerisation (irradiation with gamma-rays) of pig mucosal heparin have been characterised by mono- and two-dimensional NMR spectroscopy. Integration of typical 1H- and 13C-NMR signals provided a useful quantification of their sulfation pattern. The availability of the corresponding parent heparins showed that the original structure (including that of the active site for antithrombin, as also confirmed by affinity chromatography) had not been significantly modified by the depolymerisation procedure. This process involves only a slight decrease of undersulfated sequences. A peculiarity of gamma-LMWH is the absence of the 'linkage region', commonly present in unmodified heparins and most LMWHs.
From the leaf surface exudate of the aerial parts of Salvia cinnabarina a new secoisopimarane diterpenoid with a non-specific spasmolytic activity on histamine-, acetylcholine-, and barium chloride-induced contractions in the isolated guinea-pig ileum was obtained. The IC50 value obtained was comparable with that obtained for papaverine. The structure of 3,4-secoisopimara-4(18),7,15-triene-3-oic acid was established by 1D and 2D NMR spectroscopic techniques.
The sulfation patterns of pig and bovine mucosal commercial heparin preparations can be characterized and distinguished from each other easily by analysis of their monodimensional proton and carbon nuclear magnetic resonance ((1)H and (13)C-NMR) spectra. NMR spectroscopy can detect and quantify signals associated with major sequences as well as with minor residues such as the typical ones associated with the antithrombin (AT) binding sequence and the "linkage region." Contaminants arising from industrial preparation processes are also detectable.
A complex of the synthetic tetrasaccharide AGA*IM [GlcN, 6-SO3-alpha(1-4)-GlcA-beta(1-4)-GlcN,3, 6-SO3-alpha(1-4)-IdoA-alphaOMe] and the plasma protein antithrombin has been studied by NMR spectroscopy. 1H and 13C chemical shifts, three-bond proton-proton (3JH-H) and one-bond proton-carbon coupling constants (1JC-H) as well as transferred NOEs and rotating frame Overhauser effects (ROEs) were monitored as a function of the protein : ligand molar ratio and temperature. Considerable changes were observed at both 20 : 1 and 10 : 1 ratios (AGA*IM : antithrombin) in 1H as well as 13C chemical shifts. The largest changes in 1H chemical shifts, and the linewidths, were found for proton resonances (A1, A2, A6, A6', A1*, A2*, A3*, A4*) in GlcN, 6-SO3 and GlcN,3,6-SO3 units, indicating that both glucosamine residues are strongly involved in the binding process. The changes in the linewidths in the IdoA residue were considerably smaller than those in other residues, suggesting that the IdoA unit experienced different internal dynamics during the binding process. This observation was supported by measurements of 3JH-H and 1JC-H. The magnitude of the three-bond proton-proton couplings (3JH1-H2 = 2.51 Hz and 3JH4-H5 = 2.23 Hz) indicate that in the free state an equilibrium exists between 1C4 and 2S0 conformers in the ratio of approximately 75 : 25. The chair form appears the more favourable in the presence of antithrombin, as inferred from the magnitude of the coupling constants. In addition, two-dimensional NOESY and ROESY experiments in the free ligand, as well as transferred NOESY and ROESY spectra of the complex, were measured and interpreted using full relaxation and conformational exchange matrix analysis. The theoretical NOEs were computed using the geometry of the tetrasaccharide found in a Monte Carlo conformational search, and the three-dimensional structures of AGA*IM in both free and bound forms were derived. All monitored NMR variables, 1H and 13C chemical shifts, 1JC-H couplings and transferred NOEs, indicated that the changes in conformation at the glycosidic linkage GlcN, 6-SO3-alpha(1-4)-GlcA were induced by the presence of antithrombin and suggested that the receptor selected a conformer different from that in the free state. Such changes are compatible with the two-step model [Desai, U.R., Petitou, M., Bjork, I. & Olson, S. (1998) J. Biol. Chem. 273, 7478-7487] for the interaction of heparin-derived oligosaccharides with antithrombin, but with a minor extension: in the first step a low-affinity recognition complex between ligand and receptor is formed, accompanied by a conformational change in the tetrasaccharide, possibly creating a complementary three-dimensional structure to fit the protein-binding site. During the second step, as observed in a structurally similar pentasaccharide [Skinner, R., Abrahams, J.-P., Whisstock, J.C., Lesk, A.M., Carrell, R.W. & Wardell, M.R. (1997) J. Mol. Biol. 266, 601-609; Jin, L., Abrahams, J.-P., Skinner, R., Petitou, M., Pike, R. N. & Carrell, R.W. (1997) Proc. Natl Acad. Sci. USA 94, 14683-14688], conformational changes in the binding site of the protein result in a latent conformation.
An identification system for aquatic bacteria which would be useful for aspects ranging from pollution monitoring to health care, is described. It contains relevant information concerning the nature, taxonomy and activity of aquatic bacteria and allows to identify fresh isolates by means of their chemotaxonomic profiles. The system is simple and user friendly, so that it can be used by people not familiar with computers and has a modular interface that allows an easy interaction with laboratory instruments. The system is also connected with artificial neural network based programs to identify strains starting from the considered chemotaxonomic features.
The sequence (IdoA2SO3-GalNAc4SO3)n contributes to the HCII-mediated inhibition of thrombin by dermatan sulfate (DS). This sequence clearly results from the 13C NMR spectrum and can be quantified by the signal C1-H of IdoA2SO3 in the 1H NMR spectrum. A linear correlation has been found between the content in the disulfated disaccharide delta Di-2,4diS obtained by enzymatic demolition with ABC lyase, the percentage content in IdoA2SO3 quantified by 1H NMR, and the HCII-mediated activity of dermatan sulfates from beef mucosa and pig skin. DSs have been obtained also from pig mucosa and contain an amount, not negligible, of delta Di-4, 6diS. This disulfate disaccharide contributes to the activity expressed by the IdoA2SO3-GalNAc4SO3 sequence. The analytical techniques HPLC and 1H NMR, applied to the currently performed analyses of DS, are described and discussed.
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Dermatan sulphate (DS) obtained from bovine and pig mucosa and pig skin, and charge-enriched fractions of a selected DS preparation, were characterized in terms of charge density, M(r) and disaccharide composition of chondroitin ABC lyase digests, and by 13C-n.m.r. spectroscopy. Besides the major IdoA-GalNAc4SO3 sequences, all DS preparations contain about 10% disulphated disaccharide sequences (mostly IdoA2SO3-GalNAc4SO3, with minor amounts of IdoA-GalNAc4,6SO3). DS fragments (prepared by radical-catalysed depolymerization of DS and retaining the internal structure of the parent polysaccharide) as well as Smith degraded fragments [SD-DS, obtained by controlled degradation of periodate-oxidized and borohydride-reduced DS (RO-DS)] with the general structure GalNAc4SO3(IdoA2SO3-GalNAc4SO3)n-R (where R is the remnant of a glycol-split uronic acid, and n = 2-3 and 3-4) were characterized by one- and two-dimensional 1H-n.m.r., 13C-n.m.r. and disaccharide composition analysis. In accordance with previous findings [Maimone and Tollefsen (1990) J. Biol. Chem. 265, 18263-18271], only fragments with n > or = 3 significantly enhance the heparin cofactor II-mediated inhibition of thrombin. In natural DS preparations and their fractions, this activity (as well as the antithrombotic activity in an animal model) appears to require IdoA2SO3-containing sequences. The heparin cofactor II activity of DS, RO-DS and SD-DS fragments decreases with decreasing M(r). However, RO-DS fragments are more active than DS fragments of similar M(r), probably because of the extra flexibility endowed by glycol-split IdoA residues.
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Aggregation of platelets, stimulated by different agonists, was inhibited by omitting sample stirring or by preincubation of platelets with a monoclonal antibody against glycoproteins IIb-IIIa or with a pentapeptide containing the sequence Arg-Gly-Asp-Ser. In platelets stimulated by collagen, ADP and epinephrine, the inhibition of aggregation paralleled a reduction of both release reaction and thromboxane A2 formation. When thrombin was the stimulus, ATP release and thromboxane A2 production were unaffected (or only slightly modified) by the inhibition of platelet aggregation. These data add further evidence to the hypothesis that aggregation supports the activation of platelets stimulated by weak agonists.
We studied the influence of the occupancy of the fibrinogen receptor (GP IIb-IIIa complex) on two early aspects of agonist induced platelet activation: the increase of the intracellular Ca2+ concentration and the cytoskeleton reorganization. A monoclonal antibody, a peptide containing the RGD sequence and fibrinogen purified from human plasma were used as GP IIb-IIIa ligands. The obtained results demonstrated that fibrinogen receptor occupancy inhibits Ca2+ movement and cytoskeleton reorganization caused by low thrombin concentration and ADP.
Platelet aggregation in whole blood (WB) and in platelet rich plasma (PRP) was studied in 18 consecutive patients affected by idiopathic myelofibrosis (IM). On the basis of WB studies, 22% of patients were classified as normo-aggregating and 22% as hypo-aggregating, while 55% had spontaneous platelet aggregation (SPA). SPA was observed also when platelets from patients were stirred in the presence of normal erythrocytes, while it never occurred when normal platelets were stirred in the presence of red cells from patients. PRP studies revealed that 33% and 66% of patients were, respectively, hypo- and normo-aggregating. The most frequent abnormality in PRP was represented by defective or absent aggregation response to epinephrine.
A young women affected by Hodgkin's disease developed chronic autoimmune thrombocytopenic purpura. Splenectomy induced normalization of her platelet count, but hemorrhagic symptoms did not disappear. The patient's platelets did not aggregate in response to collagen and ADP and the IgG fraction of the patient's plasma induced the same defect in normal platelets. The women's IgG recognized glycoproteins IIb and IIIa of normal platelet membranes. Prednisone therapy induced the disappearance of bleeding symptoms and the normalization of platelet aggregation.
A murine monoclonal antibody against glycoproteins IIb-IIIa of platelet membrane completely abolished platelet aggregation induced by epinephrine, arachidonic acid, and low concentration of collagen and thrombin, but it had only minor inhibitory effects on aggregation induced by ADP, higher amounts of collagen and thrombin, and these agents combined in pairs. The simultaneous studies of aggregation and ATP secretion demonstrated that aggregation plays an essential role in stimulating the platelet-release reaction, except for the thrombin-induced ATP secretion that seems to be largely dependent on fibrinogen binding to platelet surface.
Thrombin treated with phenylmethanesulphonyl fluoride, like active enzyme, promotes modifications to human platelet cytoskeleton. The removal of active thrombin by hirudin partially reverses this process. Chymotrypsin-treated platelets do not modify their cytoskeleton after thrombin stimulus, but are still able to increase their adhesiveness to collagen. It is concluded that thrombin influences the cytoskeleton and adhesion by non-enzymic mechanisms which may be mediated by different modulators.