[Factors influencing the choice of nondonor families in a French organ-harvesting center]. / Analyse des raisons motivant le refus du don d'organes par les familles de patients en état de mort encéphalique dans un centre régional de prélèvement.

OBJECTIVES: Report the reasons that lead families to refuse organ donation during their close solicitation by hospital coordination. MATERIAL AND METHODS: A retrospective study was conducted between 2012 and 2015, including 148 (34%) refusal of organ donation among 426 patients identified in a state of brain death. A questionnaire of the family was completed for each interview. Collected data concerned patient characteristics, cause of death, description of the interview and reasons for refusal. A descriptive statistical analysis was performed. RESULTS: The median age of patients was 50 years with a sex ratio of 1.4 men to 1 woman. The most common reason for non-donor family was the desire to maintain the integrity of the body of the patient (28%) followed by a religious order pattern (11%), brutality and suddenness of death (9%), the denial of death (6%) and early age of the donor (5%). In 39% of cases, the family said that the donor had expressed a written or oral refusal in his lifetime. CONCLUSION: A better understanding of the reasons leading to the refusal of non-donor family could provide assistance to the medical team on actions to general public with the aim to reduce the refusal rate. LEVEL OF EVIDENCE: 4.

Development of a novel workflow method to characterize and validate clinical data quality acquired through the Veterinary Committee on Trauma registry.

OBJECTIVE: To generate and apply a novel workflow method to assess the quality of data from the Veterinary Committee on Trauma (VetCOT) registry. ANIMALS: Canine and feline trauma patient data entered by identified and verified Veterinary Trauma Centers into the VetCOT registry between April 2017-December 2018 were retrieved for analysis. METHODS: Analysis software (RVetQual) was created in the R programming language to compare 5,000 cases exported from the VetCOT registry with samples of original corresponding records from 6 veterinary trauma centers. In addition, an evaluation of the consistency and completeness of the trauma registry was conducted. RESULTS: The utilization of this analysis tool allowed an assessment of the VetCOT trauma registry. Some of the variables effecting the accuracy, consistency, and completeness of the VetCOT trauma registry were canine and feline age, weight, trauma time entered, and mismatches in blood glucose. However, the completeness of the database was minimally affected. CLINICAL RELEVANCE: RVetQual is an efficient, accessible, and adjustable tool that facilitates the assessment of the data quality of the VetCOT registry. Such an assessment can lead to improvement of the quality of information serving to guide further trauma patient care.

Acute insulin response to arginine in deceased donors predicts the outcome of human islet isolation.

Despite a stringent donor selection, human islet isolation remains frustratingly unpredictable. In this study, we measured acute insulin response to arginine (AIRarg), an in vivo surrogate measure of islet mass, in 29 human deceased donors before organ donation, and correlated values with the outcome of islet isolation. Thirteen isolations (45%) met the threshold for clinical islet transplantation. Among all measured donor characteristics, the only discriminating variable between successful or unsuccessful isolations was donor AIRarg (p < 0.01). Using a threshold of 55 microIU/mL (ROC curve AUC: 72%), isolation was successful in 12/19 donors with high AIRarg and in 1/10 donors with low AIRarg (p < 0.001). The negative and positive predictive values were 90 and 63%, respectively. If used to select donors in the entire cohort, AIRarg would have increased our success rate by 40% and avoided 56% of unsuccessful isolations while missing only 8% of successful preparations. Our results suggest that donor AIRarg is markedly superior to body mass index (BMI) and other criteria currently used to predict isolation outcome. If routinely performed in deceased donors, this simple test could significantly reduce the failure rate of human islet isolation.

The role of spiral CT for the assessment of the intracranial circulation in suspected brain-death.

BACKGROUND AND PURPOSE: The aim of this study was to assess the role of spiral CT for the diagnosis of brain death. METHODS: Over a 12-month period, 15 patients that fulfilled the clinical criteria of brain death were referred from the intensive care unit to evaluate remaining intracranial blood flow by spiral CT. The clinical diagnosis was confirmed by an apnea test in all cases. Two phases of spiral CT were performed at 20 and 60 seconds after the start of contrast media injection. Qualitative analysis included the evaluation of vessel opacification (arteries and veins) by two radiologists in consensus. RESULTS: The cortical segments of the middle cerebral artery (MCA) were assessable in all patients, whereas the internal cerebral veins could not be evaluated in five patients due to artifacts or intracranial hemorrhage. Opacification of the major branches of the circle of Willis was observed in seven cases. Unilateral opacification of cortical branches of the MCA occurred in one. We did not observe bilateral enhancement of cortical MCA branches. The internal cerebral veins did not enhance in brain death. CONCLUSION: The absence of internal cerebral vein opacification and the absence of bilateral enhancement of cortical MCA branches constituted the best criteria of brain death by contrast enhanced spiral CT.

Aggregation properties of beta-galactosidase of human urine and degradation of its natural substrates by a purified preparation of the enzyme.

Acid beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was purified to near homogeneity from normal human urine by two affinity chromatography steps. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the major protein band had an apparent molecular weight of 59000, thus being 5000 daltons smaller than the protein purified from human liver. Upon gel filtration on Sephadex G-150 column the purified enzyme had an apparent molecular weight of 70000 of pH 7.0. At pH 4.0 partial aggregation to a dimer of an apparent molecular weight of 150000 was found. Addition of 0.1 M galactose caused at pH 3.5, but not at pH 4.0 and 7.0, an increased formation of multimeric beta-galactosidase which eluted with the void volume of the column. Crude beta-galactosidase from human urine showed a higher aggregation tendency than the purified enzyme. None of the conditions produced an enzyme species of an apparent molecular weight of less than 40000. pH-activity profiles were measured against p-nitrophenyl-beta-D-galactoside, 3H-labelled GM1-ganglioside, [3H]keratan sulfate and the pentasaccharide O-beta-(1 leads to 4)-[6-3H]galactopyranosyl-O-beta-(1 leads to 2)-2-deoxy-2-acetamidoglycopyranosyl-O-alpha-(1 leads to 6)-mannopyranosyl-O-beta-(1 leads to 4)-mannopyranosyl-2-deoxy-2-acetamidoglucopyranoside. While p-nitrophenyl-beta-D-galactopyranoside and GM1-ganglioside were optimally hydrolyzed at pH 4.0, keratan sulfate and the pentasaccharide were optimally degraded at pH 4.3 and pH 5.0, respectively. With the chromogenic substrate and with GM1-ganglioside Km values of 0.33 mM were calculated. At pH 3.5 the hydrolysis of the synthetic substrate did not follow Michaelis-Menten kinetics. Two enzyme species appeared with Km values of 0.006 mM and 3.2 mM, respectively. The affinity of beta-galactosidase for [3H]keratan sulfate and the 3H-labelled pentasaccharide was at least one order of magnitude lower than for the amphiphilic substrates. Keratan sulfate and GM1-ganglioside did not act as competitive inhibitors of p-nitrophenyl-beta-galactosidase at the concentration tested. These findings could be explained by the existence of different binding sites for the substrates used.

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man.

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.

2-Acetamidoglucal, a new metabolite isolated from the urine of a patient with sialuria.

A new metabolite, namely 2-acetamidoglucal, has been found in the urine of a patient with sialuria in addition to the metabolites N-acetylneuraminic acid, N-acetylmannosamine, N-acetylglucosamine and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reported earlier. the structure has been identified by mass spectrometry and 360 MHz proton nuclear magnetic resonance spectroscopy and verified by synthesis. All accumulated compounds fit into the metabolic pathway for the biosynthesis of CMP-N-acetylneuraminic acid. Sialuria is discussed in terms of a failure of regulation of UDP-N-acetylglucosamine 2-epimerase.

Structure of the three major sialyl-oligosaccharides excreted in the urine of five patients with three distinct inborn diseases: "I cell disease" and two new types of mucolipidosis.

The urine of five patients with three distinct diseases ("I Cell disease" and two new types of mucolipidosis) contains sialic acid-rich oligosaccharides in a high amount: 50- to 500-fold the normal. The structure of the major components are as follows: alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNac(1 leads to 2)alphaMan(1 leads to 3)betaMan(1 leads to 4)GlcNac,[alphaAcNeu(2 leads to 6)]betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 3)[betaGal(1 leads to 4)betaGlcNac(1 leads to 2)alphaMan(1 leads to 6)]betaMan(1 leads to 4)GlcNAc and alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 3)[alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 6)]betaMan(1 leads to 4)GlcNAc. These results suggest that a deficit in alpha-neuraminidase is associated to these three different disorders and that an endo-beta-D-N-acetylglucosaminidase is able to release sialyoligosaccharides by splitting the sialylglycans of glycoproteins.

Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus.

The seed lectin (DBL) from the leguminous plant Dolichos biflorus has a unique specificity among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipitation of blood group A+H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal) containing pentasaccharide, and about 40 times better by the Forssman disaccharide (GalNAc(alpha1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL-Forssman disaccharide complex.A comparison with the binding sites of Gal-binding legume lectins indicates that the low affinity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher affinity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104. Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the first structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an alpha-helix sandwiched between two monomers. In both oligomers, this alpha-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel quaternary structure, related to the quaternary structure of the DBL heterotetramer, and brings the number of know legume lectin dimer types to four.

A reexamination of the role of GABA in the mammalian suprachiasmatic nucleus.

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that gamma-amino butyric acid (GABA)A receptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath- or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.

Structure of three Kdn-containing oligosaccharide-alditols released from oviducal secretions of Ambystoma tigrinum: characterization of the carbohydrate sequence Fuc (alpha 1-5) [Fuc (alpha 1-4)] Kdn (alpha 2-3/6).

Oligosaccharide-alditols released by reductive beta-elimination from the egg jelly coat of Ambystoma tigrinum were analyzed by 1H NMR spectroscopy. As observed for five other amphibian species, these carbohydrate chains are highly species-specific, and should support the species-specificity of gamete interaction. The carbohydrate chains of Ambystoma tigrinum are characterized by the presence of a new type of sequence: Fuc (alpha 1-5) [(Fuc (alpha 1-4)] Kdn (alpha 2-3/6).

Definitive chemical evidence for the constitutive ability of Candida albicans serotype A strains to synthesize beta-1,2 linked oligomannosides containing up to 14 mannose residues.

We have previously reported the presence of phosphate bound beta-1,2 linked oligomannosides with unusually high degrees of polymerization (DP > 7) in the mannan of Candida albicans strain VW32. To confirm this observation, we have prepared these oligomannosides from the mannan of C. albicans strain NIH A 207. Gel filtration chromatography and TLC analysis revealed DP up to 14. For both strains, NMR analysis confirmed the exclusive presence of beta-1,2 linkages in the pools of oligomannosides with a DP higher than 6 which presented an average DP of 10.6 (VW32) and 10.4 (NIH A 207). These results are important to consider in relation with the ability of these C. albicans derived oligomannosides to trigger TNFalpha synthesis according to their DP.

Carbohydrate structures of hen ovomucoid. A mass spectrometric analysis.

The apparently homogenous N-glycosidically-linked glycans 1, 7, 11 and 14 released by hydrazinolysis from hen ovomucoid were analysed by fast atom bombardment and electron-impact mass spectrometry after reduction and permethylation. The spectra support the primary structures established independently [FEBS Letters (1983) 152, 145-152] using methylation analysis, partial acid hydrolysis and 500 MHz 1H NMR spectroscopy. In addition to the major constituents present in fractions 1, 7, 11 and 14, four minor components not detected by other methods could be characterized with the aid of the mass spectrometry data as: Man2GlcNAcGlcNAc-o1, GlcNAc4Man3GlcNAc-o1, GlcNAc6Man3GlcNAc-o1 and GalGlcNAc6-Man3GlcNAc-o1. Our results show that the physical techniques used provide valuable data on the structure of complex glycans. In addition they can be employed to ascertain the homogeneity of the compounds examined as well as to detect trace amounts of homologs that might not be noticed by other methods.

Characterization of Le(x), Le(y) and A Le(y) antigen determinants in KDN-containing O-linked glycan chains from Pleurodeles waltlii jelly coat eggs.

Novel acidic oligosaccharides were isolated in large amounts by reductive alkaline treatment of the jelly coat of Pleurodeles waltlii (Michah) eggs. The oligosaccharides were found to contain the newly described KDN as acidic monosaccharide and possess either the Le(x), Le(y) and A Le(y) antigenic determinants. Occurrence of Le(x) and Le(y) determinants previously recognized as tumor-associated antigen (TAA) demonstrates that mucins of lower animals may represent a rich and easily available source for preparing TAA. Moreover, it reinforces the hypothesis according to which TAA are evolution markers.

Primary structure of a novel N-glycosidic carbohydrate unit, derived from hen ovomucoid. A 500-MHz 1H-NMR study.

The N-glycosidic carbohydrate chains of hen beta-ovomucoid were released from the protein by hydrazinolysis, and separated by HPLC. Primary structural analysis of 3 major fractions was conducted by applying 500-MHz 1H-NMR spectroscopy in combination with methylation analysis. One of the fractions investigated appeared to consist of an intersected penta-antennary structure extended with one Gal residue. The location of the latter in a certain branch could be established unambiguously by NMR. This structure is a novel member of the family of N-glycosidic carbohydrates of glycoproteins.

Analytical and comparative chromatographic maps of oligosaccharides produced by acetolysis and partial acid hydrolysis of glycoprotids.

The authors describe a chromatographic mapping procedure of oligosaccharides present in acetolysates and partial acid hydrolysates of glycopeptides or glycoproteins. Oligosaccharides are first fractionated on charcoal-Celite columns and then identified by paper chromatography. This procedure is sensitive and reproducible and allows to compare the structure of N-glycans and of glycoproteins from various sources.

Structure of the three major fucosyl-glycoasparagines accumulating in the urine of a patient with fucosidosis.

Fifteen fucosyl-oligosaccharides and fucosyl-glycoasparagines have been isolated from the urine of a patient with fucosidosis. The structure of the three most abundant glycoasparagines are as follows: alpha-Fuc-(1 lead to 6)-beta-GlcNAc-Asn; alpha-Man-(1 leads to 6)-beta-Man-(1 leads to 4)-beta-GlcNAc-(1 lead to 4) [alpha-Fuc-(1 leads to 6)]-belta-GlcNAc-Asn; beta-Gal-(1 leads to 4) [alpha-Fuc-(1 lead to 3)] beta-GlcNAc-(1 leads to 2)-alpha-Man-(1 lead to 6)-beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4) [alpha-Fuc-(1 leads to 6)] beta-GlcNAc-Asn. The structures are related to the class of fucosyl-glycoproteins (e.g.: IgG immunoglobulin, lactotransferrin and alpha 1-acid glycoprotein). The terminal sequence: beta-Gal-(1 leads to 4) [alph-Fuc-(1 leads to 3)] beta-GlcNAc-(1 leads to 2)-a-Man leads to R is novel for carbohydrate moieties in glycoproteins.

Lysosomal catabolic pathway of N-glycosylprotein glycans.

In vitro study of the initiation steps of catabolism of N-glycosylproteins in rat liver lysosomes has led to the evidence that the degradation of the carbohydrate chain is an ordered and bi-directional phenomenon: 1) The first one starts at the reducing terminus, immediately follows the degradation of the peptidic backbone by proteases and involves a serial reaction of 3 enzymes, respectively, 1) an alpha-L-fucosidase, 2) an aspartylglucosaminidase, and 3) an acidic 'oligosaccharide specific' endo-N-acetyl-beta-D-hexosaminidase, that we propose calling endo-chitobiase. 2) The second one is the commonly admitted sequential recurrent degradation by exoglycosidases. This process explains the presence of oligosaccharides sharing a single terminal reducing N-acetylglucosamine residue in human lysosomal storage diseases. Using a model glycoasparagine as a substrate, we followed the above mentioned hydrolysis reactions by 1H NMR spectroscopy. The kinetic data revealed a rapid hydrolysis of the substrate via pathway 1 prior to the action of exoglycosidases. Moreover, the latter act at different rates on the different antennae of the substrate.

Characterization of a novel endo-N-acetyl-beta-D-glucosaminidase from the culture filtrate of a basidiomycete (Sporotricum dimorphosporum), active on biantennary mono- and asialoglycoasparagines of the N-acetyllactosaminic type.

A novel endo-N-acetyl-beta-D-glucosaminidase has been characterized in a culture filtrate from a Basidiomycete. This enzyme hydrolyses biantennary monosialo and asialo-glycoasparagines of the N-acetyllactosaminic type. Endo-N-acetyl-beta-D-glucosaminidase from the Basidiomycete released from different glycoasparagines, beta-GlcNAc-(1 leads to 4)-N[14C] acetyl-Asn, alpha Fuc-(1 leads to 6)-beta-GlcNAc-(1 leads to 4)-N-[14C]acetyl-Asn and oligosaccharides which have been separated by paper and thin-layer chromatography. Determination of the radioactivity of the labeled fragments leads to the conclusion that the biantennary glycoasparagines of the N-acetyllactosaminic type are hydrolyzed after 1 h incubation at 37 degrees, to the extent of 15 per cent for the monosialo; 90 per cent for the asialo; 21 per cent for the asialo-monofucosylated, and 15 per cent for the asialo-difucosylated glycans. Tri- and tetraantennary asialo-glycans of the N-acetyllactosaminic type are not hydrolyzed by this enzyme.

[Study of the fucose-rich oligosaccharides in the urine of healthy and melituric subjects with A, B and O blood groups]. / Etude des oligosaccharides riches en fucose présents dans les urines de sujets sains et mélituriques de groupes sanguins A, B et O

The application of adsorption chromatography on charcoal-Celite leads the authors to characterize in normal urines a class of fucose-rich oligosaccharides which possess blood group activities and are related to the phenotypes ABH, Le and secretor. Most of these oligosaccharides have a glucose residue in reducing terminal positions. Excretion of some oligosaccharides increases in the urine of diabetic and lactosuric subjects. In spontaneous or induced galactosurias, the elimination of oligosaccharides with a glucose residue in reducing terminal position decreases while appears a large amount of new oligosaccharides which all possess a galactose residue in reducing terminal position. These results lead to the conclusion that urinary oligosaccharides do not originate from glycosphingolipids, but from transglycosylation on carbohydrates which exist free in the organism: glucose for normal and diabetic subjects, lactose or galactose for lactosuric and galactosuric subjects, respectively.