A Universal Photochemical Method to Prepare Carbohydrate Sensors Based on Perfluorophenylazide Modified Polydopamine for Study of Carbohydrate-Lectin Interactions by QCM Biosensor

A universal photochemical method to prepare carbohydrate sensors based on perfluorophenylazide (PFPA) modified polydopamine (PDA) for the study of carbohydrate–lectin interactions by a quartz crystal microbalance (QCM) biosensor was developed. The PFPA was immobilized on PDA-coated gold sensors via Schiff base reactions. Upon light irradiation, the underivatized carbohydrates were inserted into the sensor surface, including mannose, galactose, fucose and N-acetylglucosamine (GlcNAc). Carbohydrate sensors were evaluated for the binding to a series of plant lectins. A kinetic study of the interactions between mannose and concanavalin A (Con A), fucose and Ulex europaeus agglutinin I (UEA-I) were performed. This method can eliminate the tedious modification of carbohydrates, improve the experimental efficiency, and reduce the experimental cost, which is of great significance for the development of QCM biosensors and the study of biomolecular interactions.

Fabrication of Carbohydrate Chips Based on Polydopamine for Real-Time Determination of Carbohydrate–Lectin Interactions by QCM Biosensor

A novel approach for preparing carbohydrate chips based on polydopamine (PDA) surface to study carbohydrate–lectin interactions by quartz crystal microbalance (QCM) biosensor instrument has been developed. The amino-carbohydrates were immobilized on PDA-coated quartz crystals via Schiff base reaction and/or Michael addition reaction. The resulting carbohydrate-chips were applied to QCM biosensor instrument with flow-through system for real-time detection of lectin–carbohydrate interactions. A series of plant lectins, including wheat germ agglutinin (WGA), concanavalin A (Con A), Ulex europaeus agglutinin I (UEA-I), soybean agglutinin (SBA), and peanut agglutinin (PNA), were evaluated for the binding to different kinds of carbohydrate chips. Clearly, the results show that the predicted lectin selectively binds to the carbohydrates, which demonstrates the applicability of the approach. Furthermore, the kinetics of the interactions between Con A and mannose, WGA and N-Acetylglucosamine were studied, respectively. This study provides an efficient approach to preparing carbohydrate chips based on PDA for the lectin–carbohydrate interactions study.

Localization of α1-2 Fucose Glycan in the Mouse Olfactory Pathway

Glycoconjugates in the olfactory system play critical roles in neuronal formation, and α1-2 fucose (α1-2Fuc) glycan mediates neurite outgrowth and synaptic plasticity. Histochemical findings of α1-2Fuc glycan in the mouse olfactory system detected using Ulex europaeus agglutinin-I (UEA-I) vary. This study histochemically assessed the main olfactory and vomeronasal pathways in male and female ICR and C57BL/6J mice aged 3-4 months using UEA-I. Ulex europaeus agglutinin-I reacted with most receptor cells arranged mainly at the basal region of the olfactory epithelium. The olfactory nerve layer and glomerular layer of the main olfactory bulb were speckled with positive UEA-I staining, and positive fibers were scattered from the glomerular to the internal plexiform layer. The lateral olfactory tract and rostral migratory stream were also positive for UEA-I. We identified superficial short-axon cells, interneurons of the external plexiform layer, external, middle and internal tufted cells, mitral cells and granule cells as the origins of the UEA-I-positive fibers in the main olfactory bulb. The anterior olfactory nucleus, anterior piriform cortex and olfactory tubercle were negative for UEA-I. Most receptor cells in the vomeronasal epithelium and most glomeruli of the accessory olfactory bulb were positive for UEA-I. Our findings indicated that α1-2Fuc glycan is located within the primary and secondary, but not the ternary, pathways of the main olfactory system, in local circuits of the main olfactory bulb and within the primary, but not secondary, pathway of the vomeronasal system.

Lectin Histochemical Study of Testicular Spermatogenic Cells in Muntjak (Muntiacus muntjak muntjak)

The objective of this study was to identify the type of specific glycoconjugates and its distribution in testicular spermatogenic cells in muntjak (Muntiacus muntjak muntjak) based on lectins histochemistry. An adult male muntjak aged 4-5 years old in hard antler period was used in this study. Testicular tissue was fixed in Bouin solution and processed histologically. Histochemistry method was performed using six types biotinylated lectins such as peanut agglutinin (PNA), soybean agglutinin (SBA), wheat germ agglutinin (WGA), ricinus communis agglutinin (RCA), concanavalin A (Con A), and ulex europaeus agglutinin I (UEA I) with 20 µg/ml of concentration for PNA lectins and 15µg/ml for other type of lectins. The results showed that glycoconjugates were detected by all type of lectins except UEA I in testicular spermatogenic cells with variation in distribution pattern and also the intensity of lectins binding. Glycoconjugates β-galactose, β-glucose, mannose, Nacetylgalactosamine, N-acetylglucosamine and sialic acid were stained intensely by lectins in golgy-cap phase and acrosomal phase of spermatids. Glycoconjugate N-acetylgalactosamine was the sugar residues which distributed abundantly that marked by positive reaction with PNA, SBA, and RCA lectins. In conclusion, glycoconjugates are detected in testicular spermatids cells of muntjak indicated that glycoconjugates have an important role in spermatogenesis particularly in spermiogenesis. Key words: glycoconjugates, lectins, spermatid, spermatozoa, muntjak

Glycocalyx characterisation and glycoprotein expression of Sus domesticus epididymal sperm surface samples

The sperm surface is covered with a dense coating of carbohydrate-rich molecules. Many of these molecules are involved in the acquisition of fertilising ability. In the present study, eight lectins (i.e. Arachis hypogae (peanut) agglutinin (PNA), Lens culimaris (lentil) agglutinin-A (LCA), Pisum sativum (pea) agglutin (PSA), Triticum vulgari (wheat) germ agglutinin (WGA), Helix pomatia agglutinin (HPA), Phaseolus vulgaris (red kidney bean) leucoagglutinin (PHA-L), Glycine max (soybean) agglutinin (SBA) and Ulex europaeus agglutinin I (UEA-I)) were investigated to identify changes in the nature and localisation of glycoproteins in boar spermatozoa migrating along the epididymal duct. Complementary procedures included measurement of global lectin binding over the surface of the viable sperm population by flow cytometry, analysis of lectin localisation on the membrane of individual spermatozoa using fluorescence microscopy and the electrophoretic characterisation of the major sperm surface glycoprotein receptors involved in lectin binding. A significant increase was found in sperm galactose, glucose/mannose and N-acetyl-d-glucosamine residues distally in the epididymis. Moreover, the sperm head, cytoplasmic droplet and midpiece were recognised by most of the lectins tested, whereas only HPA and WGA bound to the principal piece and end piece of the sperm tail. Fourteen sperm surface proteins were observed with different patterns of lectin expression between epididymal regions. The sperm glycocalyx modifications observed in the present study provide an insight into the molecular modifications associated with epididymal maturation, which may be correlated with the degree of maturation of ejaculated spermatozoa.

Abstract 1945: Levels Of Circulating Progenitor Cells In Patients With Chronic Heart Failure Predict Outcomes

Background: A low level of circulating endothelial progenitor cells (EPCs), a marker of reduced vascular regeneration, is related to worse outcome in patients with coronary heart disease, but little is known on pts with heart failure (HF). The aim was to assess the prognostic value of circulating EPCs in patients with HF. Methods: In 68 pts enrolled to GISSI-HF trial by 5 Italian Centers and 45 pts from Frankfurt, mononuclear cells from peripheral blood were isolated and plated; after 4 days, adherent cells were incubated with DiI-labeled acetylated LDL, stained with FITC-labeled Ulex europaeus agglutinin I (lectin), and counted as double positive at confocal microscopy. 66 patients had ischemic etiology, 12 were females, 26 had NYHA class >II, median age 64 y (Q1–Q3 56–70y). Cutoffs for EPCs were assessed by ROC. Results: Mean EPCs in pts with HF 34 ± 7 EPCs/mm 2 , 2–3 fold lower than in age-matched controls. Over a median of 2.5 years, 12 CV deaths and 47 first hospitalizations for CV reasons were recorded. Levels of EPCs were unrelated to severity of HF, age or sex. After adjustment for clinical variables, creatinine and BNP, EPCs <30/mm 2 were associated with a 2-fold higher risk of CV death and hospitalization, as shown by K-M curves (figure ) and by Cox multivariate regression. Pts with HF of ischemic etiology gave identical results. Conclusions : The level of circulating EPCs is an independent predictor of CV death and hospitalization in pts with chronic HF. The assay of this cellular marker of risk can be performed in blood samples collected in a multicenter setting under controlled conditions, and may offer an accessible tool to assess the role of vascular regeneration in the treatment of HF.