Localization of Galectins within Glycocalyx.

Galectins are involved in various biological processes, e.g. cell-cell and cell-matrix adhesion and the transmission of cellular signals. Despite the diversity of functions, little is known about the nature of their physiological cognate ligands on the cell surface and the localization of galectins in the glycocalyx, although this information is important for understanding the functional activity of galectins. In this work, localization of endogenous and exogenously loaded galectins in the glycocalyx was studied. The following main conclusions are drawn: 1) galectins are not evenly distributed within the glycocalyx, they are accumulated in patches. Patching is not the result of a cross-linking of cellular glycans by galectins. Instead, patch-wise localization is the consequence of irregular distribution of glycans forming the glycocalyx; 2) galectins are accumulated in the inner zone of the glycocalyx rather than at its outer face or directly in vicinity of the cell membrane; 3) patches are not associated with cell rafts.

Specificity of human galectins on cell surfaces.

Galectins are ß-galactoside-binding proteins sharing homology in amino acid sequence of their carbohydrate-recognition domain. Their carbohydrate specificity outside cells has been studied previously. The main conclusion of these studies was that several levels of glycan ligand recognition exist for galectins: (i) disaccharide Galß1-4GlcNAc (LN, N-acetyllactosamine) binds stronger than ß-galactopyranose; (ii) substitution at O-2 and O-3 of galactose residue as well as core fragments ("right" from GlcNAc) provides significant increase in affinity; (iii) similarly glycosylated proteins can differ significantly in affinity to galectins. Information about the natural cellular receptors of galectins is limited. Until recently, it was impossible to study specificity of cell-bound galectins. A model based on controlled incorporation of a single protein into glycocalyx of cells and subsequent interaction of loaded cells with synthetic glycoprobes measured by flow cytometry made this possible recently. In this review, data about glycan specificity of proto-, chimera-, and tandem-repeat type galectins on the cell surface are systematized, and comparative analysis of the results with data on specificity of galectins in artificial systems was performed. The following conclusions from these studies were made: (i) cellular galectins have practically no ability to bind disaccharide LNn, but display affinity to 3'-substituted oligolactosamines and oligomers LNn; (ii) tandem-repeat type galectins recognize another disaccharide, namely Galß1-3GlcNAc (Le(c)); (iii) on the cell surface, tandem-repeat type galectins conserve the ability to display high affinity to blood group antigens of ABH system; (iv) in general, when galectins are immersed into glycocalyx, they are more selective regarding glycan interactions. Thus, we conclude that competitive interaction of galectins with cell microenvironment (endogenous cell glycans) is the main factor providing selectivity of galectins in vivo.

Human tandem-repeat-type galectins bind bacterial non-ßGal polysaccharides.

Galectins are multifunctional effectors, for example acting as regulators of cell growth via protein-glycan interactions. The observation of capacity to kill bacteria for two tandem-repeat-type galectins, which target histo-blood epitopes toward this end (Stowell et al. Nat. Med. 16:295-301, 2010), prompted us to establish an array with bacterial polysaccharides. We addressed the question whether sugar determinants other than ß-galactosides may be docking sites, using human galectins-4, -8, and -9. Positive controls with histo-blood group ABH-epitopes and the E. coli 086 polysaccharide ascertained the suitability of the set-up. Significant signal generation, depending on type of galectin and polysacchride, was obtained. Presence of cognate ß-galactoside-related epitopes within a polysaccharide chain or its branch will not automatically establish binding properties, and structural constellations lacking galactosides, like rhamnan, were found to be active. These data establish the array as valuable screening tool, giving direction to further functional and structural studies.

Treatment of cannabis dependence using escitalopram in combination with cognitive-behavior therapy: a double-blind placebo-controlled study.

BACKGROUND: Cannabis is the most frequently used illegal substance in the United States and Europe. There is a dramatic increase in the demand for treatment for cannabis dependence. Cannabis users frequently have co-morbid mood symptoms, especially depression and anxiety, and regular cannabis users may self-medicate for such symptoms. OBJECTIVES: We report a double-blind, placebo-controlled treatment study, for the prevention of cannabis use in cannabis-dependent individuals. METHOD: Regular cannabis-dependent users (n = 52) were treated for 9 weeks with weekly cognitive-behavior and motivation-enhancement therapy sessions together with escitalopram 10 mg/day. Urine samples were collected to monitor delta-9 tetrahydrocannabinol (THC) during treatment and questionnaires were administered to assess anxiety and depression. RESULTS: We observed a high rate of dropout (50%) during the 9-week treatment program. Fifty-two patients were included in the intention-to-treat analysis. Of these, ten (19%) remained abstinent after 9 weeks of treatment as indicated by negative urine samples for THC. Escitalopram provided no advantage over placebo in either abstinence rates from cannabis or anxiety and depression scores during the withdrawal and abstinent periods. CONCLUSIONS: Escitalopram treatment does not provide an additional benefit either for achieving abstinence, or for the treatment of the cannabis withdrawal syndrome. Due to limitations of our study, namely, a high dropout rate and effects of low abstinence rates on measures of anxiety, depression and withdrawal, it is premature to conclude that selective serotonin reuptake inhibitors are not effective for treatment of the cannabis withdrawal syndrome.

Integrated capture, transport, and magneto-mechanical resonant sensing of superparamagnetic microbeads using magnetic domain walls.

An integrated platform for the capture, transport, and detection of individual superparamagnetic microbeads is described for lab-on-a-chip biomedical applications. Magnetic domain walls in magnetic tracks have previously been shown to be capable of capturing and transporting individual beads through a fluid at high speeds. Here it is shown that the strong magnetostatic interaction between a bead and a domain wall leads to a distinct magneto-mechanical resonance that reflects the susceptibility and hydrodynamic size of the trapped bead. Numerical and analytical modeling is used to quantitatively explain this resonance, and the magneto-mechanical resonant response under sinusoidal drive is experimentally characterized both optically and electrically. The observed bead resonance presents a new mechanism for microbead sensing and metrology. The dual functionality of domain walls as both bead carriers and sensors is a promising platform for the development of lab-on-a-bead technologies.

Carbohydrate specificity of chicken and human tandem-repeat-type galectins-8 in composition of cells.

The network of adhesion/growth-regulatory galectins in chicken (chicken galectin, CG) has only one tandem-repeat-type protein, CG8. Using a cell-based assay and probing galectin reactivity with a panel of fluorescent neoglycoconjugates (glycoprobes), its glycan-binding profile was determined. For internal validation, human galectin-8 (HG8) was tested. In comparison to HG8, CG8 showed a rather similar specificity: both galectins displayed high affinity to blood group ABH antigens as well as to 3'-sialylated and 3'-sulfated lactosamine chains. The most remarkable difference was found to be an ability of HG8 (but not CG8) to bind the disaccharide Galß1-3GlcNAc (Le(c)) as well as branched and linear oligolactosamines. The glycan-binding profile was shown to be influenced by glycocalix of the cell, where the galectin is anchored. Particularly, glycosidase treatment of galectin-loaded cells led to the change of the profile. Thus, we suppose the involvement of cis-glycans in the interaction of cell-anchored galectins with external glycoconjugates.

Galectins promote the interaction of influenza virus with its target cell.

Influenza virus is known to bind sialoglycans located on the surface of the host cell. In addition, recent data suggest the involvement of other molecular targets in viral reception. Of note, a high density of terminal galactose residues is created on the surface of virions because of the influenza virus' own neuraminidase activity. Thus, we suggested the possibility for an interaction of the influenza virus with galactose-binding proteins--galectins. In the present work we studied the influence of several galectins on the adhesion and further internalization of virus into the cell; six virus strains and three cell lines were studied. Chicken galectins CG-1A and -2 as well as human galectins HGal-1 and -8 promote virus binding in dose dependent manner, but they do not influence the internalization stage. Also, galectins are able to restore the ability of influenza virus to infect desialylated cells up to the level of native cells. When CG-1A in physiological concentrations was loaded onto viruses, the adhesion level was higher than in the case of on-cell loading. The effect of adhesion increase depends on the glycan structure of target-cell as well as of virus. The aggregated data suggest a promotional effect of galectins during the stage of influenza virus binding with the surface of target-cell.

Solid-phase assays for study of carbohydrate specificity of galectins.

We have recently shown that the carbohydrate-binding pattern of galectins in cells differs from that determined in artificial (non-cellular) test-systems. To understand the observed discrepancy, we compared several test-systems differing in the mode of galectin presentation on solid phase. The most representative system was an assay where the binding of galectin (human galectins-1 and -3 were studied) to asialofetuin immobilized on solid phase was inhibited by polyacrylamide glycoconjugates, Glyc-PAA. This approach permits us to range quantitatively glycans (Glyc) by their affinity to galectin, i.e. to study both high and low affinity ligands. Our attempts to imitate the cell system by solid-phase assay were not successful. In the cell system galectin binds glycoconjugates by one carbohydrate-recognizing domain (CRD), and after that the binding to the remaining non-bound CRD is studied by means of fluorescein-labeled Glyc-PAA. In an "imitation" variant when galectins are loaded on adsorbed asialofetuin or Glyc-PAA followed by revealing of binding by the second Glyc-PAA, the interaction was not observed or glycans were ordered poorly, unlike in the inhibitory assay. When galectins were adsorbed on corresponding antibodies (when all CRDs were free for recognition by carbohydrate), a good concentration dependence was observed and patterns of specificities were similar (though not identical) for the two methods; notably, this system does not reflect the situation in the cell. Besides the above-mentioned, other variants of solid-phase analysis of galectin specificity were tested. The results elucidate the mechanism and consequence of galectin CRD cis-masking on cell surface.

Mammalian galectins: structure, carbohydrate specificity, and functions.

Galectins are a family of beta-galactoside binding lectins, homological by a sequence of the carbohydrate-binding site. In this review literature data about structure and carbohydrate specificity of galectins are discussed. The role of galectins in the regulation of cell adhesion in immune response, inflammation, and cancer progression is considered.

Probing sialic acid binding Ig-like lectins (siglecs) with sulfated oligosaccharides.

Soluble siglecs-1, -4, -5, -6, -7, -8, -9, and -10 were probed with polyacrylamide glycoconjugates in which: 1) the Neu5Ac residue was substituted by a sulfate group (Su); 2) glycoconjugates contained both Su and Neu5Ac; 3) sialoglycoconjugates contained a tyrosine-O-sulfate residue. It was shown that sulfate derivatives of LacNAc did not bind siglecs-1, -4, -5, -6, -7, -8, -9, and -10; binding of 6'-O-Su-LacNAc to siglec-8 was stronger than binding of 3'SiaLacNAc. The relative affinity of 3'-O-Su-TF binding to siglecs-1, -4, and -8 was similar to that of 3'SiaTF. 3'-O-Su-Le(c) displayed two-fold weaker binding to siglec-1 and siglec-4 than 3'SiaLe(c). The interaction of soluble siglecs with sulfated oligosaccharides containing sialic acid was also studied. It was shown that siglecs-1, -4, -5, -6, -7, -9, and -10 did not interact with these compounds; binding of 6-O-Su-3'SiaLacNAc and 6-O-Su-3'SiaTF to siglec-8 was weaker than that of the corresponding sulfate-free sialoside probes. Siglec-8 displayed affinity to 6'-O-Su-LacNAc and 6'-O-Su-SiaLe(x), and defucosylation of the latter compound led to an increase in the binding. Sialoside probes containing tyrosine-O-sulfate residue did not display increased affinity to siglecs-1 and -5 compared with glycoconjugates containing only sialoside. Cell-bound siglecs-1, -5, -7, and -9 did not interact with 6-O-Su-3'SiaLacNAc, whereas the sulfate-free probe 3'SiaLacNAc demonstrated binding. In contrast, the presence of sulfate in 6-O-Su-6'SiaLacNAc did not affect binding of the sialoside probe to siglecs. 6'-O-Su-SiaLe(x) displayed affinity to cell-bound siglecs-1 and -5; its isomer 6-O-Su-SiaLe(x) bound more strongly to siglecs-1, -5, and -9 than SiaLe(x).

Search for additional influenza virus to cell interactions.

Sialyl oligosaccharides have long been considered to be the sole receptors for influenza virus. However, according to [1] some viruses are able to grow in sialic-free MDCK cells. Here we attempted to reveal a possible second, non-sialic receptor, hypothesizing the involvement of additional carbohydrate lectin recognition in influenza virus reception process, first of all in situations when a lectin of the host cell could recognize the viral carbohydrate ligand. We tested the presence of galactose- and sialic acid-binding lectins, as well as mannoside- and sulfo-N-acetyllactosamine-recognizing properties of MDCK and Vero cells using polyacrylamide neoglycoconjugates and antibodies. MDCK cells bind galactoside probes stronger than Vero cells, whereas Vero cells bind preferentially sialoside, mannoside and various sulfo-oligosaccharide probes. The probing of viruses with the neoglycoconjugates revealed specific 6'-HSO (3) LacNAc (but not other sulfated oligosaccharides) binding property of A and B human strains. Affinity of 6'-HSO (3) LacNAc probe was comparable with affinity of 6'-SiaLac probe but the binding was not inhibited by the sialooligosaccharide.

Galectins as markers of aggressiveness of mouse mammary carcinoma: towards a lectin target therapy of human breast cancer.

Galectins, beta-galactoside binding proteins, expressed selectively in human breast carcinoma are attractive targets to employ lectin-aimed therapeutics. We examined beta-galactoside binding potency of neoplastic cells using fluorescein-labelled synthetic glycoconjugates as probes for flow cytometry. As a result, surface beta-galactoside binding proteins/galectins were discovered on mouse mammary carcinoma cells in vitro and in vivo unlike non-malignant cells from the several tissues; and asialo-GM1 ganglioside carbohydrate part--containing probe was the most specific one. However, in liver and lung metastatic cells galectins seem to be expressed within cytoplasm and/or nuclei. Galectin expression correlated directly with aggressive tumour potential in the A/Sn transplantable model similar to findings in several human breast carcinoma cell lines. However, galectin expression was reduced during tumour progression in more aggressive forms of spontaneous BLRB mammary carcinomas like it was shown for human breast carcinoma specimens. Analysis of the histopathological data led, however, to the conclusion that galectin expression hardly might be a suitable marker of aggressiveness of heterogeneous mammary carcinomas as the observed level of galectin expression is influenced by the amount of the stroma in a tumour sample and/or probably, galectin expression inversely correlates with tumour aggressiveness during the initial and advanced steps of mammary tumour progression. We conclude that surface beta-galactoside binding proteins/galectins that are selectively expressed during mouse mammary carcinoma progression, similarly to human breast carcinomas, seem to be proper targets for asialo-GM1-vectored cytotoxics and our mouse model system might be a relevant instrument to further test novel modes of anti-breast cancer therapy.

Sialoside-binding macrophage lectins in phagocytosis of apoptotic bodies.

Elimination of apoptotic bodies is one of the important functions of macrophages. The aim of this work was to study the role of macrophage lectins in this process. Macrophage lectins were probed with neoglycoconjugates Glyc-PAA-fluo where carbohydrate is linked to fluorescein-labeled polyacrylamide (MW 30 kD). It was shown that neoglycoconjugates containing a Neu5Acalpha2-3Gal fragment bound to macrophages isolated from blood of healthy donors. Besides, carbohydrate chains containing the same fragment were revealed on apoptotic bodies. Phagocytosis of apoptotic bodies by macrophages was inhibited with sialooligosaccharide ligands of siglec-5 and MAbs to siglec-5. Thus, siglec-5 expressed on macrophages could participate in phagocytosis of apoptotic bodies. In addition, the role of siglecs in engulfment of apoptotic bodies by tumor-associated macrophages was studied. The phagocytic potency of macrophages isolated from blood of breast cancer patients was lower than engulfment ability of macrophages obtained from healthy donors and depended on tumor degree. Staining of macrophages obtained from blood of tumor patients with sialylated Glyc-PAA-fluo probes was more intense than that of macrophages from healthy donors; phagocytosis of apoptotic bodies by tumor-associated macrophages was inhibited by carbohydrates that are known to be ligands for siglecs.

Induction of differentiation in leukemic cell strains with myelopeptide-4.

We studied the capacity of myelopeptide-4 (regulatory peptide of the bone marrow origin) to induce terminal differentiation of HL-60 and K-562 leukemic cells. Myelopeptide-4 increased the expression of CD14 and CD38 differentiation antigens on the surface of HL-60 cells and of CD44 antigen on K-562 cells, induced the appearance of mature monocyte/macrophages in HL-60 culture and hemoglobin-producing cells in K-562 cell culture, and stimulated phagocytic activity of THP-1 leukemic cells. Myelopeptide-4 is an endogenous factor of cell differentiation, a prospective agent for antileukemic therapy.

The role of TNF-alpha in amygdala kindled rats.

In the present study, the interaction between epileptogenesis and the immune system were studied in a kindling model. First, the effects of a single administration of TNF-alpha (5.0 microg/kg, i.p.) on seizure and EEG activity were investigated in amygdala-kindled rats. TNF-alpha treated rats showed more prolonged epileptiformic discharges than control rats. TNF-alpha also induced a decrease in the power of delta band and an increase in theta and alpha activity. In addition, a marked increase in the power of beta and gamma band was observed. The EEG changes were most numerous in the frontal cortex and amygdala. All effects were registered 24 h after TNF-alpha administration. Finally, electrical stimulation enhanced the level of TNF-alpha in blood serum from 1.9 +/- 1.5 to 12.7 +/- 3.8 pg/ml and in brain tissue 56.8 +/- 6.0 to 109.2 +/- 6.0 pg/mg, as was determined via the ELISA method. It can be concluded that there is a mutual facilitative interaction of both epileptogenic and cytokine-derived mechanisms on this type of seizure. The changes in the power spectrum of the EEG after TNF-alpha might contribute to intensify thalamic-derived facilitation of epileptic discharge in cortical structures.

Fluorescent carbohydrate probes for cell lectins.

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe(x)-PAA-flu, Sia2-PAA-flu, GlcNAc2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe(x)-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe(x)-PAA-3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe(x)-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

Cellular localization of the galactose-binding lectin from human serum.

An SL2 lectin was isolated from human serum and characterized previously; cellular localization of the lectin was studied using polyclonal rabbit antibodies. According to cytofluorimetry, anti-SL2 antibodies bound only to lymphocytes and monocytes but not to other blood cells. Antibodies bound to Jurkat T cell lymphoma but did not interact with IM-9 cells of B cell origin. Moreover, the Jurkat cells bound oligosaccharides having the highest affinity to SL2 (GalNAcalpha_and Fucalpha1-2Gal), and this interaction was inhibited by anti-SL2 antibodies. Lysis of the Jurkat cells with subsequent electrophoresis and Western blotting indicates that anti-SL2 antibodies recognized a 14-kD protein.

A chromosomal region of Mycoplasma agalactiae containing vsp-related genes undergoes in vivo rearrangement in naturally infected animals.

A Mycoplasma agalactiae genomic fragment carrying four vsp-related genes (designated avg: agalactiae variable genes) was cloned, sequenced and compared to the vspA gene of Mycoplasma bovis. The following features were revealed: (i) the presence of a highly conserved vsp 5' upstream region; (ii) a highly homologous vsp N-terminal end encoding a putative lipoprotein signal sequence; (iii) sequence divergence of the rest of the mature proteins. By using avg specific probes in Southern blot analysis of genomic DNAs of M. agalactiae strains as well as of isolates from infected animals, marked DNA polymorphism of avg fragments was demonstrated. In addition, the avg genomic fingerprints were monitored for a period of 7 months, in isolates of M. agalactiae from an individual chronically infected animal. The results provided evidence that a chromosomal region of M. agalactiae, carrying vsp-related genes, undergoes rearrangements in vivo in the natural animal host during the course of infection.

Glycosylation alterations of cells in late phase apoptosis from colon carcinomas.

Comparisons of carbohydrate profiles between control and apoptotic colon carcinoma cells were performed by flow cytometry using a set of lectins and anti-carbohydrate antibodies. The six cell lines analyzed presented distinct carbohydrate profiles before induction of apoptosis. PHA-L and MAA binding decreased after induction of apoptosis by UV-treatment. In contrast an increase of PNA binding was observed after induction of apoptosis, except on SW-48 cells for which a decrease occurred. A decrease of SNA binding was observed after induction of apoptosis from strongly positive control cell lines, whereas it increased on weakly positive ones. All the blood group related antigens A, H, Lewis a, Lewis x, Lewis b, and Lewis y, had their expression strongly diminished on apoptotic cells. These changes occurred irrespective of the mode of apoptosis induction since similar results were obtained after UV, TNFalpha, or anti-Fas treatment. Fucosyltransferases activities were also decreased after apoptosis induction, except for alpha1,3fucosyltransferase in anti-Fas treated HT-29 cells, where it was strongly augmented. This could be attributed to the IFNgamma preteatment required to induce Fas expression on these cells. Fucosidase activity decreased after induction of apoptosis suggesting that it was not responsible for the loss of fucosylated structures. In the rat PRO cell line, H blood group antigens are mainly carried by a high molecular weight variant of CD44. It could be shown that the loss of H antigen after induction of apoptosis correlated with a loss of the carrier glycoprotein.