Epidemiology of the vascular assessment and correlation of the WIfI Classification in lower limb amputee patients at Martinique university hospital in 2018.

BACKGROUND: The precise epidemiological evaluation of amputations is difficult. It is a serious public health and economic problem with a high death rate. The proportion of amputees with pre-amputation vascular status remains unknown. The main objective of our study was to evaluate the proportion of patients with lower limb amputation who had a pre-procedural vascular assessment. The secondary objectives were to evaluate the risk of amputation at the admission of these patients, estimate the incidence of amputations in Martinique, and to collect epidemiological data on this category of patients. MATERIAL AND METHODS: We conducted an epidemiological, retrospective, and observational study, over the year 2018 between January 01 and December 31, including all adults' patients who underwent an amputation of the lower limb at the university hospital center of Martinique. RESULTS: Among the 170 included patients, 79 (46%) patients had a major lower limb amputation. The incidence of amputations in 2018 was estimated at 48.9/100,000 inhabitants. The vascular assessment was performed for 110 (65%) patients. For the other 60 (35%) patients who did not have a vascular assessment, 53 (88%) had a severe infection. This assessment was significantly related to the amputation level: a vascular assessment was performed in 97 (70%) patients with below the knee amputation versus 13 (41%) patients with above the knee amputation (P<0.01). The WIfI classification system found a high risk of amputation for 152 (89%) of patients but also a benefit of revascularization ranked high for 138 (81%) of them. The origin of amputation was limb ischemia for 125 (68%) patients. CONCLUSION: A significant number of patients who underwent lower limb amputation did not have a pre-procedural vascular assessment. Many improvements in the health care are therefore to be implemented. The upcoming M@diCICAT project in Martinique will contribute in the improvement of patient management. The incidence of amputation in Martinique is considered high compared to other countries (French national incidence in 2003=24.8/100,000 inhabitants), and it seems to have remained stable since 2008. Our population is considered to be at high risk of amputation by the SVS-WIfI classification. This score seems adapted to anticipate the evolution of these patients and could be useful in daily practice.

Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols.

The MUC1 glycoprotein, epitectin, a component of the human bladder epithelium, was purified from human urine. Sedimentation equilibrium analysis and gel filtration using polysaccharide or protein standards revealed a polydisperse preparation with molecular weights ranging from about 0.9 to 1.3 x 10(6). This suggests that in the native state epitectin exists as aggregates of three or four monomer units of 350-400 kDa. Epitectin was found to have significant affinity to hexyl-, octyl- or phenyl agarose indicating that hydrophobic interactions and possibly carbohydrate-carbohydrate interactions may be responsible for the self-association. Chemical and enzymic deglycosylation of [125I]-labeled urine epitectin and metabolically labeled H.Ep.2 epitectin resulted in extremely polydisperse products. The buoyant densities of epitectin purified from urine and H.Ep.2 cells were found to be 1.39-1.40 g ml(-1), suggesting that the total carbohydrate content of these preparations is not significantly different. The O-linked saccharides of epitectin were fractionated by HPLC and analyzed by permethylation and FAB-MS. The neutral saccharides from both sources contain three common structures, namely Gal1 --> 3GalNAc, GlcNAc1 --> 6 (Gal1 --> 3) GalNAc and Gal1 --> 4GlcNAc --> 6 (Gal1 --> 3)GalNAc. The sialic acid of urine epitectin consisted entirely of N-acetylneuraminic acid. The two sources of epitectin, in vitro labeled on sialic acid, were found to have the same sialyl oligosaccharides but in different proportions. Metabolic labeling and N-glycanase susceptibility experiments firmly established the presence of N-linked saccharides in epitectin as minor components. The remarkable similarities in the total carbohydrate content, the carbohydrate composition and structures of saccharides between epitectin from urine, a non-malignant source, and H.Ep.2 cells is surprising in view of the prevailing view that MUC1 glycoproteins of cancer cells are underglycosylated compared to those produced by non-malignant cells.

Structure determination of a novel uronic acid residue isolated from the exopolysaccharide produced by a bacterium originating from deep sea hydrothermal vents.

The exopolysaccharide produced by the bacterium Alteromonas sp. strain 1644 originating from deep sea hydrothermal vents was shown to contain a novel glucuronic acid derivatives: 3-O-[(R)-1-carboxyethyl]-D-glucuronic acid. The structure of this compound was established on the basis of mass spectrometric data, methylation analysis, preparation of derivatives, and chemical synthesis of references compounds.

The glycan moiety of human pancreatic lithostathine. Structure characterization and possible pathophysiological implications.

Lithostathine, also known as pancreatic stone protein, pancreatic thread protein or regenerating protein, is a glycoprotein which is normally found in the exocrine pancreas, whereas in other tissues it appears either only under pathological conditions, such as Alzheimer's disease (brain), cancer (colon) or during regeneration (endocrine pancreas). In the latter case, it has been shown recently that it acts as a growth factor which stimulates islet regeneration. Little is known about its glycan moiety, which conceivably might be involved in this tissue specificity and pathophysiological characteristics. Therefore we isolated the major oligosaccharide chains of human pancreatic lithostathine and determined their sequences by means of NMR analysis. We obtained eleven different glycoforms and we were able to determine the sequence of seven of them. They all were from the same site of glycosylation (Thr5) and displayed the same core 2 structure: GlcNAc(beta 1-6)[Gal(beta 1-3)]GalNAc alpha-. They ranged in size from 4 to 9 sugar residues. Elongation was found to proceed from a common tetrasaccharidic core: Gal(beta 1-4)GlcNAc(beta 1-6)[Gal(beta 1-3)]GalNAc-ol through N-acetyllactosamine units. The non-reducing ends of some oligosaccharides carry the antigenic determinant H, with presence of external Fuc linked only in (alpha 1-2) to Gal. All the glycans, except one, carry a sialic acid in (alpha 2-3) linkage to Gal, with one disialylated form which displays a supplementary (alpha 2-6) linkage. These findings are consistent with the polymorphism of the protein, shown by means of SDS gel electrophoresis and isoelectric focusing, either in its native form or after enzymic processing. Moreover, sialylation seems to protect to some extent the Arg11-Ile12 bond from in situ hydrolysis, thus preventing the harmful precipitation of the C-terminal polypeptide in the pancreatic ducts.

Chemical and immunological characterization of the extracellular galactomannan of Aspergillus fumigatus.

The galactomannan (GM) produced extracellularly by Aspergillus fumigatus has been purified by a double sequential hydrazine-nitrous acid treatment of the ethanol precipitate of the culture filtrate. Nuclear magnetic resonance and gas-liquid chromatography-mass spectrometry analysis have been performed on intact GM, acid-hydrolyzed GM, and oligomers resulting from the acetolysis of the acid-hydrolyzed GM. Results show that A. fumigatus GM is composed of a linear mannan core with an alpha-(1-2)-linked mannotetraose repeating unit attached via alpha-(1-6) linkage. Side chains composed of an average of 4 to 5 beta-(1-5)-galactofuranose units are linked to C-6 and C-3 positions of alpha-(1-2)-linked mannose units of the mannan. The immunoreactivity of GM and HCl-hydrolyzed GM was studied by use of human sera from aspergillosis patients and an antigalactofuran monoclonal antibody. The alpha-(1-2) (1-6)-mannan core is not antigenic. The immunogenic galactofuran is found amongst several exocellular glycoproteins. According to a direct enzyme-linked immunosorbent assay with GM as the detector antigen, only 26% of the serum samples from aspergilloma patients (all positive by immunodiffusion assays) give optical density values superior to a cutoff estimated as the mean +/- 3 standard deviations of values obtained with control sera.

Production and Characterization of an Exopolysaccharide Excreted by a Deep-Sea Hydrothermal Vent Bacterium Isolated from the Polychaete Annelid Alvinella pompejana.

The heterotrophic and mesophilic marine bacterium HYD-1545 was isolated on a metal-amended medium from the dorsal integument of the hydrothermal vent polychaete Alvinella pompejana. This strain, which can be assigned to the genus Alteromonas on the basis of its G+C content and phenotypical features, produced large amounts of an acidic polysaccharide in batch cultures. The polysaccharide was excreted during the stationary phase of growth and contained glucose, galactose, glucuronic acid, galacturonic acid, and 4,6-O-(1-carboxyethilidene)-galactose as major components. This polysaccharide was a polyelectrolyte, and the viscosity of its solutions depended on the ionic strength. The decrease in viscosity with increasing NaCl concentrations and the effect of Ca in decreasing the viscosity at low Ca concentrations support a model in which the polysaccharide carries anionic groups. However, an unusual behavior was observed at higher concentrations and could be related to intermolecular interactions involving Ca ions.

Periplasmic glucans of Pseudomonas syringae pv. syringae.

We report the initial characterization of glucans present in the periplasmic space of Pseudomonas syringae pv. syringae (strain R32). These compounds were found to be neutral, unsubstituted, and composed solely of glucose. Their size ranges from 6 to 13 glucose units/mol. Linkage studies and nuclear magnetic resonance analyses demonstrated that the glucans are linked by beta-1,2 and beta-1,6 glycosidic bonds. In contrast to the periplasmic glucans found in other plant pathogenic bacteria, the glucans of P. syringae pv. syringae are not cyclic but are highly branched structures. Acetolysis studies demonstrated that the backbone consists of beta-1,2-linked glucose units to which the branches are attached by beta-1,6 linkages. These periplasmic glucans were more abundant when the osmolarity of the growth medium was lower. Thus, P. syringae pv. syringae appears to synthesize periplasmic glucans in response to the osmolarity of the medium. The structural characteristics of these glucans are very similar to the membrane-derived oligosaccharides of Escherichia coli, apart from the neutral character, which contrasts with the highly anionic E. coli membrane-derived oligosaccharides.

Isolation of a 32 kDa Mycobacterium tuberculosis protein by lectin affinity chromatography.

A 32 kDa antigen from delipidated M. tuberculosis H37Rv culture filtrate protein extract (CFPE) was purified by affinity chromatography on immobilized Lens culinaris lectin and electroelution. This antigen represents 0.4% of the total CFPE carbohydrate content and possesses galactose, xylose, mannose and GlcNAc (5:2:3:1 mol. ratio). A monoclonal antibody against the purified antigen reacted with the 32 kDa as well as a 30 kDa antigen in H37Rv CFPE, thus suggesting that both antigens represent closely related allelomorphic forms of the same antigen.

Chondroitin sulphate covalently cross-links the three polypeptide chains of inter-alpha-trypsin inhibitor.

Inter-alpha-trypsin inhibitor (ITI) is a tight complex of three different proteins: bikunin and two heavy chains H1 and H2. In order to demonstrate that the three chains are covalently linked by a chondroitin sulphate chain as previously proposed [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherford, S. and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751], ITI was extensively digested with thermolysin and the glycosaminoglycan-containing fragment was isolated from the digest by ion-exchange chromatography. Its peptide structural determination and mass spectrometry analysis both provide evidence that the different peptide chains constituting ITI are associated by the new cross-link described as the protein-glycosaminoglycan-protein cross-link.

Bacterial clearance in mice assessed by a conductance method is enhanced by RU 41740 (Trade name Biostim®).

Conductance measurements were used to determine the bacterial concentrations in the blood of mice receiving a suspension of Escherichia coli by the intravenous route. The study was also carried out on animals that had been treated with a drug (RU 41740) that stimulates the reticuloendothelial system. The intensity of the in vivo phagocytosis was assessed by calculating the difference between the mean detection times of the treated and control groups (ΔDT). The results obtained by conductance measurements were not statistically different from those obtained by the standard method of counting on Petri dishes. The conductance measurement method was also compared with the carbon clearance test, the reference test for studying in vivo phagocytosis. The two clearance tests revealed a major stimulation of the reticuloendothelial system by RU 41740. The coefficient of variation was lowest in the case of the bacterial clearance test.

Isolation and characterisation of hemicelluloses from sunflower hulls.

The hemicelluloses extracted from sunflower hulls by repeated alternating oxidative and alkaline treatments were purified by precipitation with Cetavlon and then ion-exchange chromatography on DEAE-trisacryl. The resulting fractions were examined by hydrolysis, methylation, GLC-MS, and NMR spectroscopy. The hemicelluloses are of the glucuronoxylan type with the following structure -->4)-beta-D-Xylp-(1-->4)-[4-O-Me-alpha-D-GlcpA-(1-->2)]-bet a-D-Xylp-(1-->. The polysaccharides differed in the amount of branching; the ratio of the main fraction 4-O-MeGlcA:Xyl was 1:8-9.

Collision-induced dissociation of alkali metal cationized and permethylated oligosaccharides: Influence of the collision energy and of the collision gas for the assignment of linkage position.

Tandem mass spectrometry has been used to study the collision-induced decomposition of [M+Na](+) ions of permethylated oligosaccharides. It is shown that many linkage positions in one compound may be determined by the presence or absence, in a single spectrum, of specific fragment ions that arise from the cleavage of two ring bonds and that the yield of such ions depends strongly on the collision energy and nature of the collision gas. In contrast to the behavior of monolithiated native oligosaccharides, the collision-induced decomposition of the sodiated and permethylated oligosaccharide samples at low energy leads to preferential cleavage of glycosidic linkages. At high collision energies, the fragment ions formed by cleavage of more than one bond are greatly enhanced, especially when helium is replaced by argon as the collision gas. Furthermore, argon is the more efficient collision gas in inducing fragmentation of the precursor ions. As an example of the application of this method, the discrimination between the 1 → 3 and 1 → 6-linked mannose residues in the common core of N-glycans is described.

Structural characterization of a novel class of glycophosphosphingolipids from the protozoan Leptomonas samueli.

Aqueous phenol extraction of the lower trypanosomatid Leptomonas samueli released into the aqueous layer a chloroform/methanol/water-soluble glycophosphosphingolipid fraction. Alkaline degradation and purification by gel filtration chromatography resulted in a tetrasaccharide (phosphatidylinositol (PI)-oligosaccharide A), and a pentasaccharide (PI-oligosaccharide B), each containing 2 mol of 2-aminoethylphosphonate and 1 mol of phosphate. Nuclear magnetic resonance spectroscopy and fast atom bombardment-mass spectrometry suggested that the structure of PI-oligosaccharide A is [formula: see text] and that of PI-oligosaccharide B is as shown. [formula: see text] Both compounds contain an inositol unit linked to ceramide via a phosphodiester bridge. The major aliphatic components of the ceramide portion are stearic acid, lignoceric acid, and C20-phytosphingosine. These novel glycolipids fall within the glycosylated phosphatidylinositol (GPI) family, since they contain the core structure Man alpha (1-->4)GlcNH2 alpha (1-->6)myo-inositol-1-PO4, which is also found in the glycoinositolphospholipids and lipophosphoglycan of Leishmania spp., the L. major promastigote surface protease, the glycosylphosphatidylinositol anchor of Trypanosoma brucei variant surface glycoprotein, and the lipopeptidophosphoglycan of Trypanosoma cruzi. The glycophosphosphingolipids of Leptomonas have features in common with the glycolipids of both Leishmania and T. cruzi, resembling the former by the alpha (1-->3) linkage of mannose to the GPI core, while the 2-aminoethylphosphonate substituent on O-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Thus these data lend some support to the hypothesis that both T. cruzi and Leishmania evolved from a Leptomonas-like ancestor.

Decreased faecal exoglycosidase activities identify a subset of patients with active Crohn's disease.

1. alpha 1-Proteinase inhibitor (alpha 1-antitrypsin) is excreted in a deglycosylated form (M(r) 38,000) in the faeces of healthy subjects and in patients with quiescent Crohn's disease. By contrast, in most patients with active Crohn's disease, alpha 1-proteinase inhibitor is excreted in a glycosylated form (M(r) 51,000). 2. Faecal extracts containing deglycosylated alpha 1-proteinase inhibitor are able to deglycosylate alpha 1-proteinase inhibitor by an exoglycosidic process. Conversely, we demonstrate that in faecal extracts from patients excreting glycosylated alpha 1-proteinase inhibitor, glycosidase activities, such as N-acetyl-beta-glucosaminidase (EC 3.2.1.30), alpha-mannosidase (EC 3.2.1.24) and particularly beta-galactosidase (EC 3.2.1.23), are strongly decreased. 3. Degradation of glycosidases by proteases could not explain the decreased glycosidase activity in these faecal extracts. 4. Our data suggest that a modification of the bacterial colonic flora (or of its metabolic activity) occurs in most patients with active Crohn's disease and could be responsible for an impaired colonic salvage of carbohydrates.

The heavy chains of human plasma inter-alpha-trypsin inhibitor: their isolation, their identification by electrophoresis and partial sequencing. Differential reactivity with concanavalin A.

Inter-alpha-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as H1 and H2, respectively. We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H3 inside pre-alpha-trypsin inhibitor (Enghild et al. (1991) J. Biol. Chem. 266, 747-751). Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type N-glycans in both H1 and H2 and that of O-glycans in H2. H1 is eluted from Con-A Sepharose by alpha-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates. The capacity of H2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI.

Oligosaccharide structures of mucins secreted by the human colonic cancer cell line CL.16E.

Cl.16E, a stably differentiated clonal derivative of the human colonic cancer cell line HT29, was used to investigate the structure of oligosaccharide chains of mucins in colonic cancer. Secretory mucins were purified by equilibrium density gradient centrifugation in CsCl. Oligosaccharide side chains were isolated after beta-elimination. Compositional analysis of oligosaccharide-alditols performed after purification by gel filtration on a Bio-gel P-6 column showed 1) that GalNAc residues were located exclusively at the reducing ends of the chains, and 2) that fucose was absent from the preparation. Oligosaccharide-alditols were separated by high performance liquid chromatography (HPLC) on quaternary amine packings into a minor neutral fraction representing about 6.5% by weight of released oligosaccharides and four acidic fractions. Two acidic fractions, namely FI and FII encompassing mono- and disialylated structures, respectively, and containing 78% of total oligosaccharide alditols, were separated by HPLC. Structural determinations were carried out using methylation analysis, 1H NMR spectroscopy, and fast atom bombardment-mass spectrometry. Twelve oligosaccharide structures were determined which ranged in size from 3 to 8 residues. These oligosaccharides were based on core types 1, 2, and 4. Elongation of oligosaccharide chains was terminated by addition of sialic acid in alpha 2-3 linkage to Gal beta 1-3R and to Gal beta 1-4R residues. The predominant structure was a hexasaccharide (fraction FII-4). This contrasts with normal colonic mucins whose oligosaccharides were previously found to be based on core 3 structures and carry sialic acids in alpha (2-6) linkage to Gal beta 1-3R, to Gal beta 1-4R, and to GalNAc alpha-R (Podolsky, D.K. (1985) J. Biol. Chem. 260, 8262-8271; Podolsky, D.K. (1985) J. Biol. Chem. 260, 15510-15515). Collectively our findings suggest that Cl.16E colon cancer cells are able to synthesize mucin oligosaccharides of gastric type whose elongation is truncated by premature sialylation.

Triterpenoid saponins from Argania spinosa.

Five new oleanane saponins named arganine A, B, D, E and F and two known saponins: arganine C and mi-saponin A were isolated from the kernel of Argania spinosa. The structures of these saponins were elucidated by using 1H NMR, 1H-1H COSY NMR, 13C NMR, FAB mass spectrometry and chemical evidence.

Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin.

Amylose-defective mutants were selected after UV mutagenesis of Chlamydomonas reinhardtii cells. Two recessive nuclear alleles of the ST-2 gene led to the disappearance not only of amylose but also of a fraction of the amylopectin. Granule-bound starch synthase activities were markedly reduced in strains carrying either st-2-1 or st-2-2, as is the case for amylose-deficient (waxy) endosperm mutants of higher plants. The main 76-kDa protein associated with the starch granule was either missing or greatly diminished in both mutants, while st-2-1-carrying strains displayed a novel 56-kDa major protein. Methylation and nuclear magnetic resonance analysis of wild-type algal storage polysaccharide revealed a structure identical to that of higher-plant starch, while amylose-defective mutants retained a modified amylopectin fraction. We thus propose that the waxy gene product conditions not only the synthesis of amylose from endosperm storage tissue in higher-plant amyloplasts but also that of amylose and a fraction of amylopectin in all starch-accumulating plastids. The nature of the ST-2 (waxy) gene product with respect to the granule-bound starch synthase activities is discussed.