BACKGROUND The cardiac myocyte t-tubular system ensures rapid, uniform cell activation and several experimental lines of evidence suggest changes in the t-tubular system and associated excitation-contraction coupling proteins may occur in heart failure. METHODS AND RESULTS The organization of t-tubules, L-type calcium channels (DHPRs), ryanodine receptors (RyRs) and contractile machinery were examined in fixed ventricular tissue samples from both normal and failing hearts (idiopathic (non-ischemic) dilated cardiomyopathy) using high resolution fluorescent imaging. Wheat germ agglutinin (WGA), Na-Ca exchanger, DHPR and caveolin-3 labels revealed a shift from a predominantly transverse orientation to oblique and axial directions in failing myocytes. In failure, dilation of peripheral t-tubules occurred and a change in the extent of protein glycosylation was evident. There was no change in the fractional area occupied by myofilaments (labeled with phalloidin) but there was a small reduction in the number of RyR clusters per unit area. The general relationship between DHPRs and RyR was not changed and RyR labeling overlapped with 51±3% of DHPR labeling in normal hearts. In longitudinal (but not transverse) sections there was an ∼30% reduction in the degree of colocalization between DHPRs and RyRs as measured by Pearson's correlation coefficient in failing hearts. CONCLUSIONS The results show that extensive remodelling of the t-tubular network and associated excitation-contraction coupling proteins occurs in failing human heart. These changes may contribute to abnormal calcium handling in heart failure. The general organization of the t-system and changes observed in failure samples have subtle differences to some animal models although the general direction of changes are generally similar.

A new approach to analyze multi-component Saturation Transfer Difference (STD) NMR spectra by combining the STD and the DOSY experiment is proposed. The resulting pulse sequence was successfully used to simplify an exemplary multi-component protein/substrate system by means of standard DOSY processing methods. Furthermore, the same experiment could be applied to calculate the ratio of saturated substrate molecules and its saturation rate in the case of competitive interactions. This ratio depends on the strength of this interaction between the substrates and the protein, so that this kind of information could be extracted from the results of our experiment.

BACKGROUND: Cancer has profound effects on gene expression, including a cell's glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones. CONTENT: In this article, we survey clinical tests that target carbohydrate modifications for diagnosing and treating cancer. We present the biological relevance of glycosylation to disease progression by highlighting the role these structures play in adhesion, signaling, and metastasis and then address current methodological approaches to biomarker discovery that capitalize on selectively capturing tumor-associated glycoforms to enrich and identify disease-related candidate analytes. Finally, we discuss emerging technologies-multiple reaction monitoring and lectin-antibody arrays-as potential tools for biomarker validation studies in pursuit of clinically useful tests. SUMMARY: The future of carbohydrate-based biomarker studies has arrived. At all stages, from discovery through verification and deployment into clinics, glycosylation should be considered a primary readout or a way of increasing the sensitivity and specificity of protein-based analyses.

Lysosome reactions were ultrastructurally analyzed using a cell-free system with inside-out cell membrane vesicles (IOVs) prepared from rat erythrocyte ghosts in an alkaline buffer and with wheat germ agglutinin-coated colloidal gold particles (WGA-CGs). The submembranous surface coat in the ghosts was depleted from the IOVs' outer surfaces. When lysosomes from rat liver were incubated with these IOVs, some of the trilaminar membranes of the lysosomes and IOVs came into close contact and formed a five-laminar structure without an intermembranous gap. In other reactions, the membranes of both structures formed one continuous trilaminar membrane along the margin of contact and ruffling five-laminar structures in other regions. Several lysosomes exhibited invaginating hollows or projections that entrapped or encircled the IOVs. Similar five-laminar structures were seen at a few points of contact between the IOVs and the hollowing or projecting membranes. In contrast, such reactions were much rarer when IOVs with reconstituted spectrins and actins on their outer surface were used. The formation of tubuliform pits with membrane-bound WGA-CGs was also observed after the incubation of lysosomes with WGA-CGs. These observations suggest that lysosomes fuse with cytoskeleton-depleted IOVs, wrap arm-like projections around them, enclose them by invagination or incorporate their membrane-bound macromolecules through the process of tubuliform invagination. Furthermore, the fusion and wrapping processes are not necessarily independent.

Stage-specific differences in wheat germ agglutinin (WGA) binding saccharides were demonstrated between the surfaces of the eggs, L1 larvae, young aduhs, and old adults of Caenorhabditis elegans. The WGA binding was to n-acetylglucosamine groups but not to terminally linked n-acetylneuraminic acids. An age-related decrease in WGA binding occurred in adults, supporting previous findings of a decrease in net negative cuticle surface charge during aging.

Abstract In the rat two major molecular variants of prolactin are recorded i.e. 23,000 M(r) and glycosylated 26,000 M(r). In order to further characterize the glycosylated 26,000 rat prolactin molecular variant, rat pituitary cell lysates were digested with several glycoen-zymes and the digestion products submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis and subsequent immunoblotting. The results were as follows: treatment with 1) neuraminidase, specific for sialic acid, yielded an M(r) decrease of the glycosidic variant from 26,000 to 24,500, 23,800, 23,000 and 22,000; 2) endo-alpha-N-acetylgalactosaminidase, which releases the disaccharide Gal (beta 1-3) GalNac from O-glycans, split 26,000 rat prolactin into a doublet of M(r) 26,000 to 25,500; and 3) mixed exoglycosidases from Turbo cornutus caused a gradual M(r) shift from 26,000 to 23,000. Affinity chromatography on wheat germ agglutinin Sepharose 6MB and soybean agglutinin agarose of rat pituitary homogenates and competitive inhibition tests showed that glycosylated rat prolactin has distinct affinity for these lectins. From the experimental data it is proposed that glycosylated rat prolactin is O-linked through threonine by the disaccharide Gal (beta 1-3) GalNac and possesses at least GalNac, and/or Gal and sialyl residues.

Due to their ability to bind specifically to certain carbohydrate sequences, lectins are a frequently used tool in cytology, histology, and glycan analysis but also offer new options for drug targeting and drug delivery systems. For these and other potential applications, it is necessary to be certain as to the carbohydrate structures interacting with the lectin. Therefore, we used glycoproteins remodeled with glycosyltransferases and glycosidases for testing specificities of lectins from Aleuria aurantia (AAL), Erythrina cristagalli (ECL), Griffonia simplicifolia (GSL I-B(4)), Helix pomatia agglutinin (HPA), Lens culinaris (LCA), Lotus tetragonolobus (LTA), peanut (Arachis hypogaeae)(PNA), Ricinus communis (RCA I), Sambucus nigra (SNA), Vicia villosa (VVA), and wheat germ (Triticum vulgaris)(WGA) as well as reactivities of anti-carbohydrate antibodies (anti-bee venom, anti-horseradish peroxidase [anti-HRP], and anti-Lewis(x)). After enzymatic remodeling, the resulting neoglycoforms display defined carbohydrate sequences and can be used, when spotted on nitrocellulose or in enzyme-linked lectinosorbent assays, to identify the sugar moieties bound by the lectins. Transferrin with its two biantennary complex N-glycans was used as scaffold for gaining diverse N-glycosidic structures, whereas fetuin was modified using glycosidases to test the specificities of lectins toward both N- and O-glycans. In addition, alpha(1)-acid glycoprotein and Schistosoma mansoni egg extract were chosen as controls for lectin interactions with fucosylated glycans (Lewis(x) and core alpha1,3-fucose). Our data complement and expand the existing knowledge about the binding specificity of a range of commercially available lectins.

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70,-33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.

Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant ( K D) of 777 +/- 93 nM for SNA binding to Neu5Acalpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.

PURPOSE: Locoregional recurrence is the most common complication after adenocarcinoma resection in the colon, despite adjuvant chemotherapy. Therapy efficacy could be improved if designed to target malignant cells by incorporating specific recognition factors in the drugs or the drug vehicles. The aim of this study was to elucidate whether the overexpression of sialic acid (SA) on colonic malignant tissues could be utilized for drug targeting by cationic polymers. MATERIALS AND METHODS: Cell lines (IEC-6, SW-480 and SW-620) and colon polyps and normal adjacent tissues harvested from dimethylhydrazine (DMH) induced rats were used as in vitro and in vivo models of different metastatic stages of colon cancer. SA expression was identified by fluorescent wheat germ agglutinin (WGA), and verified by pretreatment with neuraminidase. The role of mucus in the mucosal binding experiments was explored by pretreatment with dithiothreitol (DTT). The binding of FITC labeled cationic polymers of various degrees of cationization to normal and malignant colonic cells and tissue was measured. RESULTS: SA was overexpressed on malignant colonic cells and tissues, and its expression correlated to the metastatic stage in vitro. The binding of the cationic copolymers to the cell lines and tissues correlated with the charge density of the polymer and with the metastatic stage of the cell line. The interaction between the malignant colonic cells and tissues with the polymers was SA dependent and increased after mucus removal. CONCLUSION: Cationic polymers could be used as a targeting tool to colonic malignant epithelium, to be implemented in drug delivery and diagnosis.

Lectins are polyvalent proteins of non-immune origin with exquisite carbohydrate binding specificity making them ideal for investigation of cell surface glycoprotein and glycolipid antigens. We examined the cell surface lectin binding phenotypes of 20 UM-SCC squamous cell carcinoma cell lines established from 17 patients with head and neck cancers using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of binding. Conconavalin A (Con A) from Canavalia ensiformis and the peanut agglutinin (PNA) from Arachis hypogaea bound all SCC cell lines tested and wheat germ agglutinin (WGA) from Triticum vulgaris bound to 12 of 13 tumor cell lines. The blood group O specific lectin UEA 1 from Ulex europeus also bound to all cell lines regardless of the donor blood type. Lectins of Dolichos biflorus (DBA) and Griffonia simplicifolia (GS I-B4 or BSA I-B4) with binding specificity for glycoproteins associated with blood group A and B respectively, had reactivity that did not directly correlate with blood group antigen expression. In contrast to the other lectins in our panel which exhibited broad reactivity with SCC antigens, the BSA-II lectin from Griffonia simplicfolia,(GS II or BSA II) which has sugar binding specificity for terminal non-reducing GlcNAc, did not bind to any of the screened cell lines. Our results demonstrate a common pattern of lectin-defined carbohydrate expression on the cell surface of squamous cell carcinomas of head and neck that appears promising in defining the malignant cellular phenotype. Lectin binding profile may be useful in differentiating benign from malignant histopathology.

Association constants for the binding of methyl alpha-D-galactopyranoside (methyl alpha-D-Galp) and methyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (methyl alpha-D-GalNAcp) to three Bandeiraea simplicifolia isolectins (A4, A2B2, B4) were determined by equilibrium dialysis and fluorescence enhancement measurements. The a and B subunits appear to have approximately the same Kassoc for methyl alpha-D-Galp: 1.45 X 10(4), 1.98 X 10(4), and 2.06 X 10(4) M-1 for A4, A2B2, and B4, respectively, as determined by equilibrium dialysis. Fluorescence enhancement measurements on B4 gave an association constant of 2.07 X 10(4) M-1 for methyl alpha-D-Galp and 1.87 X 10(3) M-1 for methyl beta-D-galp. By equilibrium dialysis, we were able to detect 3.3 (theory, 4.0) methyl alpha-D-GalNAcp binding sites for A4 (Kassoc = 1.87 X 10(5) M-1), 1.9 for A2B2 (Kassoc = 1.19 X 10(5) M-1), and were unable to detect any methyl alpha-D-GalNAcp binding sites for B4. However, four very weak methyl alpha-D-GalNAc binding sites for B4 were detected by fluorescence enhancement measurement (Kassoc = 1.26 X 10(2) M-1). Thus, the A subunit has an affinity for methyl alpha-D-GalNAc 3 orders of magnitude greater than the B subunit. Precipitation and hapten inhibition data are in accord with these binding measurements. Toward guaran and type B blood group substance, all isolectins precipitated the same amount of biopolymer. However, AB3, A2B2, and A3B, which are mono-, di-, and trivalent for alpha-D-GalNAcp, were differentially precipitated by type A blood group substance which contains alpha-D-GalNAcp-end groups. A3B precipitated the most, A2B2 less, and AB3 no type A substance. These isolectins should prove useful in studies evaluating the effect of valence on lectin-cell interaction.

Three fluorescein-labeled lectins have been shown to exhibit specificity for the surface of cells in different layers of the epidermis in the newborn rat. An isolectin from seeds of Bandeiraea simplicifolia with specificity for alpha-D-galactosyl end groups labeled the basal and lower spinous cells; a lectin from Ulex europaeus exhibiting specificity for alpha-L-fucosyl units outlines the surface of spinous cells, and a second lectin from B. simplicifolia, with specificity for N-acetyl-D-glucosamine, labels the cornified cells. Appropriate blocking experiments have confirmed the specific nature of the binding.

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.

An isolectin (BS I-B4) derived from Bandeiraea simplicifolia seeds and specific for terminal alpha-D-galactopyranosyl groups was found to be cytotoxic to Swiss 3T3 mouse cells. After mutagenesis and selection with BS I-B4, a variant clonal cell line resistant to both this isolectin and the alpha-D- and beta-D-galactose-binding lectin abrin was isolated. The parental cell line showed homogeneous and noninteracting binding sites for BS I-B4, whereas the variant cells exhibited a curved plot with a reduced number of binding regions. Another lectin, BS II, which is derived from the same seeds by specific for terminal N-acetyl-D-glucosaminyl groups, was cytotoxic to the variant but not the parental cells. These results suggest a possible lesion in the biosynthesis of cell surface structures resulting in the exposure of subterminal N-acetyl-D-glucosaminyl moieties in the variant line.

AIMS To investigate the effects of the combined application of an N-acyl homoserine lactone (HSL) analog and antibiotics on biofilms of Porphyromonas gingivalis, a major pathogen of periodontal disease. METHODS AND RESULTS Antibiotics used were cefuroxime, ofloxacin and minocycline. A flow-cell model was used for biofilm formation. Samples were divided into four groups: control, analog-treated, antibiotic-treated and combined application groups. Biofilm cell survival was determined using adenosine triphosphate (ATP) bioluminescence and confocal laser microscopy (CLSM). In the combined application group, the ATP count in biofilm cells was significantly decreased compared with the antibiotic-treated group (Games-Howell test, P < 0·05). A combination of cefuroxime and the analog was most effective against the P. gingivalis biofilm. CLSM observations revealed that the proportion of dead cells was highest in the combined application group. CONCLUSIONS The combined application of the N-acyl HSL analog and antibiotics was effective at reducing the viability of P. gingivalis cells in biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY The combined application of the N-acyl HSL analog and antibiotics may be successful for eradicating infections involving bacterial biofilms, such as periodontitis.