A Novel Serum Glycobiomarker for Diagnosis and Prognosis of Cholangiocarcinoma Detected by Butea monosperma Agglutinin.

Plant lectins are widely used in medical glycosciences and glycotechnology. Many lectin-based techniques have been applied for the detection of disease-associated glycans and glycoconjugates. In this study, Butea monosperma agglutinin (BMA), a lectin purified from seeds of the medicinal plant Butea monosperma, was used for the detection of cholangiocarcinoma (CCA)-associated glycans. Expression of BMA-binding N-acetyl galactosamine/galactose (GalNAc/Gal)-associated glycan (BMAG) in CCA tissues was determined using BMA lectin histochemistry; the results showed that BMAG was undetectable in normal bile ducts and drastically increased in preneoplastic bile ducts and CCA. The study in hamsters showed that an increase of BMAG was associated with carcinogenesis of CCA. Using an in-house double BMA sandwich enzyme-linked lectin assay, BMAG was highly detected in the sera of CCA patients. The level of serum BMAG in CCA patients (N = 83) was significantly higher than non-CCA controls (N = 287) and it was applicable for diagnosis of CCA with 55.4% sensitivity, 81.9% specificity, and 76.0% accuracy. A high level of serum BMAG (≥82.5 AU/mL) was associated with unfavorable survival of CCA patients; this information suggested the potential of serum BMAG as a poor prognostic indicator of CCA. In summary, BMAG was aberrantly expressed in preneoplastic bile ducts and CCA, it was also highly detected in patient serum which potentially used as a marker for diagnosis and prognostic prediction of CCA.

Roles of glycoprotein glycosylation in the pathogenesis of an endemic osteoarthritis, Kashin­Beck disease, and effectiveness evaluation of sodium hyaluronate treatment

Background/aim: We aimed to explore the roles of glycoprotein glycosylation in the pathogenesis of Kashin­Beck disease (KBD), and evaluated the effectiveness of sodium hyaluronate treatment. Materials and methods: Blood and saliva were collected from KBD patients before and after the injection of sodium hyaluronate. Normal healthy subjects were included as controls. Saliva and serum lectin microarrays and saliva and serum microarray verifications were used to screen and confirm the differences in lectin levels among the three groups. Results: In saliva lectin microarray, bindings to Sophora japonica agglutinin (SJA), Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I), Euonymus europaeus lectin (EEL), Maackia amurensis lectin II (MAL-II), Sambucus nigra lectin (SNA), Hippeastrum hybrid lectin (HHL), and Aleuria aurantia lectin (AAL) were higher in the untreated KBD patients than in the control group. Increased levels of HHL, MAL-II, and GSL-I in the untreated KBD patients discriminated them in particular from the treated ones. Jacalin was lower in the untreated KBD patients compared to the treated KBD and control groups. In serum lectin microarray, HHL and peanut agglutinin (PNA) were increased in the untreated KBD group in comparison to the control one. AAL, Phaseolus vulgaris agglutinin (E+L) (PHA-E+L), and Psophocarpus tetragonolobus lectin I (PTL-I) were lower in the untreated KBD patients compared to the treated KBD and control groups. Hyaluronate treatment appeared to normalize SNA, AAL, and MAL-II levels in saliva, and HHL, PNA, AAL, PTL-I, and PHA-E+L levels in serum. Saliva reversed microarray verification confirmed significant differences between the groups in SNA and Jacalin, in particular for GSL-I levels, while serum reversed microarray verification indicated that HHL, PNA, and AAL levels returned to normal levels after the hyaluronate treatment. Lectin blot confirmed significant differences in HHL, AAL, and Jacalin in saliva, and HHL, PNA, PHA-E+L, and AAL in serum. Conclusion: HHL in saliva and serum may be a valuable diagnostic biomarker of KBD, and it may be used as follow-up for the hyaluronate treatment.

Selenoglycosides as Lectin Ligands: 77 Se-Edited CPMG-HSQMBC NMR Spectroscopy To Monitor Biomedically Relevant Interactions.

The fundamental importance of protein-glycan recognition calls for specific and sensitive high-resolution techniques for their detailed analysis. After the introduction of 19 F NMR spectroscopy to study the recognition of fluorinated glycans, a new 77 Se NMR spectroscopy method is presented for complementary studies of selenoglycans with optimised resolution and sensitivity, in which direct NMR spectroscopy detection on 77 Se is replaced by its indirect observation in a 2D 1 H,77 Se HSQMBC spectrum. In contrast to OH/F substitution, O/Se exchange allows the glycosidic bond to be targeted. As an example, selenodigalactoside recognition by three human galectins and a plant toxin is readily indicated by signal attenuation and line broadening in the 2D 1 H,77 Se HSQMBC spectrum, in which CPMG-INEPT long-range transfer ensures maximal detection sensitivity, clean signal phases, and reliable ligand ranking. By monitoring competitive displacement of a selenated spy ligand, the selective 77 Se NMR spectroscopy approach may also be used to screen non-selenated compounds. Finally, 1 H,77 Se CPMG-INEPT transfer allows further NMR sensors of molecular interaction to be combined with the specificity and resolution of 77 Se NMR spectroscopy.

Molecular modeling and dynamic simulations of agglutinin-like family members from Candida albicans: New insights into potential targets for the treatment of candidiasis.

Infections by Candida albicans in immune compromised patients cause significant morbidity and mortality. In the search for potential molecular targets for drug development, the family of agglutinin-like proteins (Als) in C. albicans have been identified due to numerous attributes associated with high virulence, most prominently due to their role in adherence. Here, molecular models of individual members of the Als family illustrated common and unique structure features. Additionally, dynamic simulations were performed to display regions of high mobility. The results showed variations between Als members in the fluctuation of the A1B1 protein loop, which is located at the entrance to the peptide binding cavity, suggesting that this feature may be a factor contributing to observed differences in affinities to ligands and adhesion properties. Molecular docking results further suggested that ligand affinity could be influenced by movements in the A1B1 loop. In addition, a new site was identified in Als in an area adjacent to the peptide binding cavity that could serve as a new binding site for the design of future anti-adhesion ligands that provide increased specificity inhibiting Als proteins from C. albicans.

Investigation of Cuprizone Inactivation by Temperature.

Animal models, such as cuprizone (bis-cyclohexanone oxaldihydrazone) feeding, are helpful to study experimental demyelination and remyelination in the context of diseases like multiple sclerosis. Cuprizone is a copper chelator, which when supplemented to the normal food of C57BL/6J mice in a concentration of 0.2% leads to oligodendroglial loss, subsequent microglia and astrocyte activation, resulting in demyelination. Termination of the cuprizone diet results in remyelination, promoted by newly formed mature oligodendrocytes. The exact mode of cuprizone's action is not well understood, and information about its inactivation and cleavage are still not available. The knowledge of these processes could lead to a better understanding of cuprizone's mode of action, as well as a safer handling of this toxin. We therefore performed experiments with the aim to inactivate cuprizone by thermal heating, since it was suggested in the past that cuprizone is heat sensitive. C57BL/6J mice were fed for 4 weeks with 0.2% cuprizone, either thermally pretreated (60, 80, 105, 121 °C) or not heated. In addition, primary rat oligodendrocytes, as a known selective toxic target of cuprizone, were incubated with 350 µM cuprizone solutions, which were either thermally pretreated or not. Our results demonstrate that none of the tested thermal pretreatment conditions could abrogate or restrict the toxic and demyelinating effects of cuprizone, neither in vitro nor in vivo. In conclusion, the current study rebuts the hypothesis of cuprizone as a heat-sensitive compound, as well as the assumption that heat exposure is a reason for an insufficient demyelination of cuprizone-containing pellets.

In vitro immunostimulatory properties of Abrus lectins derived peptides in tumor bearing mice.

In vitro immunostimulatory effect of Abrus lectins derived peptide fractions (AGP and ABP) was investigated in DL bearing mice. Both AGP and ABP were found to activate splenocytes and induced production of cytokines like IL-2, IFN-gamma and TNF-alpha indicating a Th1 type of immune response. Analysis of in vitro treated splenocytes by flow cytometry revealed an increase in percentage of T and B cell with high expression of activation markers (CD25(+) and CD71(+)). At the same time, expression of co-stimulatory markers was significantly high compared to tumor control. The tumor associated macrophages were able to stimulate NO production, IL-1 secretion, increased phagocytosis and decreased expression of mannose receptor. It was also observed that NK cell was activated by AGP and ABP. These results suggest that both AGP and ABP act as immunostimulants in vitro in DL bearing mice.

Multivalent carbohydrate recognition on a glycodendrimer-functionalized flow-through chip.

Dendrimers were fitted out with up to eight mannose moieties by "click" chemistry. They were subsequently attached to aluminum oxide chips via a spacer that was linked to the dendrimer core; this resulted in a microarray of glycodendrimers. Binding of the glycodendrimers to the fluorescent lectins ConA and GNA was observable in real time. In a single experiment it was possible to observe the multivalency enhancement or cluster effect in the binding event. This effect was small for ConA, in agreement with its widely spaced binding sites, whereas it was large for GNA, with its twelve much more closely spaced binding sites. The dendrimer-fitted chip represents a valuable screening tool for multivalency effects. Furthermore kinetic and thermodynamic data on binding events can be deduced. Inhibition experiments are also possible with the system as was shown for ConA with alpha-methyl mannose as the inhibitor.

[Cell surface display of Yarrowia lipolytica lipase Lip2 in Saccharomyces cerevisiae with a-agglutinin as carrier protein].

OBJECTIVE: In order to display extracellular.lipase Lip2 from Yarrowia lipolytica on the surface of yeast Saccharomyces cerevisiae for whole cell catalysts. METHODS: The mature Lip2 encoding fragment was amplified from Yarrowia lipolytica total DNA, and was inserted into the 3'terminal of AGA2 to give the plasmid pCTLIP2 for surface display of Lip2. Olive oil, tributyrin and p-nitrophenyl palmitate (pNPP) were used as substrates to measure lipase activity. Moreover, the characterization of displayed lipase and its free form was analyzed. RESULTS: The surface displayed lipase was confirmed to be active towards olive oil, tributyrin and p-nitrophenyl palmitate (pNPP), and reached its highest expression level at 182 U/g dry cell after induced by galactose for 72h. The optimum temperature of cell surface displayed Lip2 was 40 degrees C After incubated at 50 degrees C for 4h, the surface displayed lipase retained 23.2% of its full activity, improved a little compared to free Lip2. The surface displayed lipase showed a preference to medium-chain and long-chain fatty acids p-nitrophenyl esters (C8-C16). CONCLUSION: The cell surface display system based on a-agglutinin is an effective system for displaying Lip2, and the whole cell EBY100-pCTLIP2 will be probably suited to a different range of applications.

Nuclear localization of STAT5A modified with O-linked N-acetylglucosamine and early involution in the mammary gland of Hirosaki hairless rat.

Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.

[Effect of Chinese herbal medicine 1023 Recipe in blocking cancer transformation of experimental precancerous lesion and its mechanism].

OBJECTIVE: To study the effect of Chinese herbal medicine 1023 Recipe in blocking cancer transformation of experimental oral precancerous lesion and its mechanism. METHODS: We treated the experimental oral precancerous lesion in hamster's cheek pouch using 1023 Recipe (consisting of Radix Astragali, Gynostemma Pentaphyllum, Rhizoma Chuanxiong and selenium-rich green tea) for 6 weeks, and observed its effect in blocking cancer transformation, detected 2 kinds of agglutinin receptors (receptors of wheat germ agglutinin and Ricinus communis agglutinin) in the mucosa of the hamster's cheek pouch. RESULTS: The rate of cancer transformation in 1023 Recipe treated group was lower than that in the control group without treatment (P<0.05). Agglutinin receptors in the two groups were different significantly. CONCLUSION: 1023 Recipe is effective in treating hyperplasia, and can prevent its cancer transformation. The mechanism may be that 1023 Recipe can induce precancerous lesions to differentiate into normal tissues.

The Interactions of Allium sativum leaf agglutinin with a chaperonin group of unique receptor protein isolated from a bacterial endosymbiont of the mustard aphid.

The homopteran sucking insect, Lipaphis erysimi (mustard aphid) causes severe damage to various crops. This pest not only affects plants by sucking on the phloem, but it also transmits single-stranded RNA luteoviruses while feeding, which cause disease and damage in the crop. The mannose-binding Allium sativum (garlic) leaf lectin has been found to be a potent control agent of L. erysimi. The lectin receptor protein isolated from brush border membrane vesicle of insect gut was purified to determine the mechanism of lectin binding to the gut. Purified receptor was identified as an endosymbiotic chaperonin, symbionin, using liquid chromatography-tandem mass spectrometry. Symbionin from endosymbionts of other aphid species have been reported to play a significant role in virus transmission by binding to the read-through domain of the viral coat protein. To understand the molecular interactions of the said lectin and this unique symbionin molecule, the model structures of both molecules were generated using the Modeller program. The interaction was confirmed through docking of the two molecules forming a complex. A surface accessibility test of these molecules demonstrated a significant reduction in the accessibility of the complex molecule compared with that of the free symbionin molecule. This reduction in surface accessibility may have an effect on other molecular interactive processes, including "symbionin virion recognition", which is essential for such symbionin-mediated virus transmission. Thus, garlic leaf lectin provides an important component of a crop management program by controlling, on one hand, aphid attack and on the other hand, symbionin-mediated luteovirus transmission.

Comparison of the lectin-binding pattern in different human melanoma cell lines.

Glycosylation is generally altered in tumour cells in comparison with their normal counterparts. These alterations are thought to be important because they contribute to the abnormal behaviour of cancer cells. Therefore, we have comparatively analysed the glycoproteins in cell extracts from human melanoma (primary site--WM35; metastatic sites-- WM239, WM9 and A375) cell lines using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and lectin staining. The glycoprotein pattern of the WM35 line differed from that of the other cell lines in having less proteins that reacted with Sambucus nigra, Maackia amurensis and Phaseolus vulgaris agglutinins. A glycoprotein of about 70 kDa had a significantly increased reaction with Sambucus nigra agglutinin in all the cell lines from metastatic sites. In the WM9, WM239 and A375 cell lines, additional bands (160-100 kDa) were stained with Phaseolus vulgaris agglutinin, suggesting that cells from metastatic sites contain more glycoproteins with beta1-6 branches. On the other hand, only minor changes in the reaction with Galanthus nivalis agglutinin, a mannose-specific lectin, were detected. Among the proteins showing different lectin staining, one, with an apparent molecular weight of 133 kDa, was recognized by antibodies as N-cadherin. The present results suggest that in human melanoma the expression of branched and sialylated complex type N-oligosaccharides consistently increased in cells from metastatic sites, and support the view that carbohydrates are associated with the acquisition of the metastatic potential of tumour cells.

CBP70, a glycosylated nuclear lectin.

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by beta-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-l agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal beta Gal residues, Gal beta 1-3 GalNAc, Man alpha 1-3 Man, sialic acid alpha 2-6 linked to Gal or GalNAc; and sialic acid alpha 2-3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and alpha-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting.

Carbohydrate-binding proteins (plant/human lectins and autoantibodies from human serum) as mediators of release of lysozyme, elastase, and myeloperoxidase from human neutrophils.

Analysis of cell surface glycosylation not only provides information about cell properties such as their state of differentiation or histogenetic lineage. The carbohydrate chains also provide potentially functional binding sites to endogenous carbohydrate-binding proteins. This interaction can elicit consequent signalling processes. Because of the importance of neutrophils in the host defence system, we monitored the effect of the binding of such sugar receptors to their cell surface on the release of the enzymatic activities of lysozyme, elastase, and myeloperoxidase. Besides the mannose-binding lectin concanavalin A and the immunomodulatory alpha/beta-galactoside-binding lectin from Viscum album L., three preparations of human sugar receptors - beta-galactoside-binding lectin (M(r) 14 kDa) and two affinity-purified polyclonal IgG fractions from serum with the capacity to recognize alpha- or beta-galactosides, respectively - were used. Two animal lectins from chicken liver and intestine that bind beta-galactosides, as well as the lectin-like human serum amyloid P component, were included in order to assess the importance of slight differences in ligand recognition. Cytochalasin B-enhanced enzyme release was invariably seen with the two plant lectins and the chicken liver beta-galactoside-binding lectin, but the related intestinal lectin did not increase enzyme release. The mammalian homologue of these avian lectins triggered lysozyme secretion, and the lactoside-binding IgG fraction enhanced the amount of extracellular elastase activity slightly but significantly. Thus, the actual lectin, not the nominal specificity of sugar receptors, is crucial for elucidation of responses. Due to the highly stimulatory activity of the two plant lectins, neutrophils from patients with non-cancerous diseases and from patients with lung cancer were monitored for the extent of lectin-mediated enzyme release. Only the concanavalin A-mediated reactivity of the neutrophils was associated with the type of disease.

Interactions of Bowringia mildbraedii agglutinin with complex- and hybrid-type glycans.

Affinity chromatography on Bowringia mildbraedii agglutinin (BMA) Sepharose of glycopeptides confirmed a previous report using oligosaccharides (Animashaun, T. and Hughes, R. C. (1989) J. Biol. Chem. 264,4657-4663) that high affinity binding requires the sequence Man alpha 1---2 Man alpha 1----6 Man alpha 1----6 Man beta 1----4. However, moderate binding was still exhibited by structures lacking this sequence provided the oligosaccharide core sequence Man alpha 1----3[Man alpha 1----6]Man beta 1----4GlcNAc was present. This moderate binding was not affected by substitution with N-acetylglucosamine at C2 and C4, respectively, of the Man alpha 1----3 and Man beta 1----4 residues and BMA Sepharose should prove to be a useful tool for the isolation of bisected or non-bisected hybrid-type glycans.

Purification and characterization of Canavalia gladiata agglutinin.

A lectin from Japanese jack bean (Canavalia gladiata agglutinin, CGA) was purified by affinity chromatography on a maltamyl-Sepharose column. On sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis, CGA was shown to have a protein subunit with a mol. wt. of 30,000. CGA has an amino acid composition similar to that of Concanavalin A. The lectin activity of CGA could be detected not only by hemagglutination assay with trypsinized human erythrocytes but also by the binding assay with intact horseradish peroxidase. The binding method could determine CGA in a concentration ranging from 50 to 500 ng/mL. The quantitative-inhibition studies of the binding indicated that CGA has sugar-binding specificities similar to those of concanavalin A.

Influência dos aglutinantes nas características físicas de comprimidos de pó de guaraná (Paullinia cupana H.B.K.). Comparaçäo com amostras adquiridas no comércio / Influence of aglutinant agents in the physical characteristics of guarana powder tablets

Foram desenvolvidas formulaçöes de comprimento de pó de guaraná (Paullinia cupana H.B.K., Sapindaceae), visando facilitar sua administraçäo por via oral, visto que este possui sabor fracamente amargo e adstringente. Foram utilizadas goma de amido, gelatina, sorbitol, xarope simples e goma arábica como aglutinantes, analisando-se em seguida as características físicas dos comprimidos obtidos no comércio. A formulaçäo contendo xarope simples, na concentraçäo de 15,5% apresentou melhor comportameno reológico, capacidade de resistência aos choques e atrito e tempo de desagregaçäo adequado às formulaçöes sólidas de liberaçäo gástrica