Ocular injury by transient formaldehyde exposure in a rabbit eye model.

Formaldehyde (FA) is frequently used in sterilizing surgical instruments and materials. Exposure to FA is highly concerned for eye tissues. Rabbit corneal epithelial cells were examined for changes after FA exposure. Our results showed that cell survival decreased 7 days after transient 3 min exposure to more than 100 ppm FA by trypan blue staining while MTT assay detected significant decrease at 20 ppm at 24 hours observation. The decrease of cell survival rate was concentration (up to 600 ppm)- and observation time (1-7 day)- dependent. The cell number decreased after 100 ppm FA exposure for more than 10 min at 7-day observation. The FA treated cells showed increased apoptosis/necrosis and cell cycle accumulation at sub G1 phase as well as mitochondria clustering around nucleus. The in vivo rabbit eye exposure for tear production by Schirmer's test revealed that the FA-induced overproduction of tear also exhibited observation time (1-10 day)- and FA concentration (20-300 ppm for 5 min exposure)-dependent. Activated extracellular signal-regulated kinase (pERK2) in cornea explants by western blotting was reduced and increased c-Jun amino - terminal kinase (JNK) activation (pJNK) in cornea and conjunctiva was evident at 2 month after exposure to 50-200 ppm FA for 5 min. In conclusion, injury to the eye with transient exposure of up to 100 ppm FA for 3 min decreased corneal cell survival while a more sensitive MTT test detected the cell decrease at 20 ppm FA exposure. Morphology changes can be observed even at 5 ppm FA exposure for 3 min at 7 days after. The FA exposure also increased apoptotic/necrotic cells and sub-G1 phase in cell cycle. Long term effect (2 months after exposure) on the eye tissues even after the removal of FA can be observed with persistent JNK activation in cornea and conjunctiva.

Mutation in fucose synthesis gene of Klebsiella pneumoniae affects capsule composition and virulence in mice.

The emerging pathogenicity of Klebsiella pneumoniae (KP) is evident by the increasing number of clinical cases of liver abscess (LA) due to KP infection. A unique property of KP is its thick mucoid capsule. The bacterial capsule has been found to contain fucose in KP strains causing LA but not in those causing urinary tract infections. The products of the gmd and wcaG genes are responsible for converting mannose to fucose in KP. A KP strain, KpL1, which is known to have a high death rate in infected mice, was mutated by inserting an apramycin-resistance gene into the gmd. The mutant expressed genes upstream and downstream of gmd, but not gmd itself, as determined by reverse transcriptase polymerase chain reaction. The DNA mapping confirmed the disruption of the gmd gene. This mutant decreased its ability to kill infected mice and showed decreased virulence in infected HepG2 cells. Compared with wild-type KpL1, the gmd mutant lost fucose in capsular polysaccharides, increased biofilm formation and interacted more readily with macrophages. The mutant displayed morphological changes with long filament forms and less uniform sizes. The mutation also converted the serotype from K1 of wild-type to K2 and weak K3. The results indicate that disruption of the fucose synthesis gene affected the pathophysiology of this bacterium and may be related to the virulence of this KpL1 strain.

Recognition intensities of submolecular structures, mammalian glyco-structural units, ligand cluster and polyvalency in abrin-a-carbohydrate interactions.

Abrin-a is the most toxic fraction of lectins isolated from Abrus precatorius seeds and belongs to the family of type 2 ribosome inactivating proteins (RIP). This toxin may act as a defense molecule in plants against viruses, fungi and insects, where attachment of abrin-a to the exposed glycans on the surface of target cells is the crucial and initial step of its cytotoxicity. Although it has been studied for over four decades, the recognition factors involved in abrin-a-carbohydrate interaction remains to be clarified. In this study, roles of mammalian glyco-structural units, ligand clusters and polyvalency in abrin-a recognition were comprehensively analyzed by enzyme-linked lectinosorbent binding and inhibition assays. The results indicate that: (i) this toxin prefers oligosaccharides having alpha-anomer of galactose (Gal) at the non-reducing terminal than the corresponding beta-anomer; (ii) Galalpha1-3Galalpha1- (B(alpha)), Galalpha1-4Gal (E), Galbeta1-3GalNAc (T) and Galbeta1-3/4GlcNAc (I/II) related oligosaccharides were the active glyco-structural units; (iii) tri-antennary II(beta), prepared from N-glycan of asialo fetuin, played a dominant role in recognition; (iv) many high-density polyvalent I(beta)/II(beta) and E(beta) glycotopes enhanced the reactivity; (v) the carbohydrate recognition domain of abrin-a is proposed to be a combination of a small cavity type of Gal as major site and a groove type of additional one to tetrasaccharides as subsites with a preference of alpha1-3/4/6Gal, beta1-3GalNAc, beta1-3/4/6GlcNAc, beta1-4/6Glc, beta1-3DAra and beta1-4Man as subterminal sugars; (vi) size of the carbohydrate recognition domain may be as large enough to accommodate a linear pentasaccharide and complementary to Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc (gailipenta) sequence. A comparison of the recognition factors and combining sites of abrin-a with ricin, another highly toxic lectin, was also performed to further understand the differences in recognition factors between these two type 2 RIPs.

Roles of mammalian structural units, ligand cluster and polyvalency in the Abrus precatorius agglutinin and glycoprotein recognition process.

Previous studies on one of the toxic type 2 ribosome inactivating proteins (RIP), Abrus precatorius agglutinin (APA), have shown that the recognition domains of APA are restricted to monomers of Galbeta1-3GalNAc (T, Thomsen-Friedenreich glycotope) and Galbeta1-3/4GlcNAc (blood group precursor type I/II sequences); which are essential but play a minor role in the recognition process. In this study, APA recognition factors were expanded to include ligand clusters and polyvalent glycotopes by enzyme-linked lectinosorbent binding and inhibition assays. Based on the results of molar relative potency, the essential mammalian structural units are Galbeta1-3GalNAcalpha/beta1- (T(alpha)/T(beta))>Galalpha1-4Gal (E)>Galbeta1-3/4GlcNAc (I/II) and avidity for tri-/di-antennary II(beta), T, E and II monomers was found to be 7.1 x 10(2), 4.0, 5.5, 3.7 and 2.4 times higher than monomeric Gal. Among natural polyvalent glycotopes or clusters, high-density polyvalent T(alpha) and complex multivalent I(beta)/II(beta) glycotopes greatly enhanced the affinity for APA over 10(4) times. Based on these results, it is concluded that contribution of monomeric T(alpha), II(beta), I(beta), E(beta) and their clusters and polyvalency play critical roles in this recognition process. The binding intensities of these factors in decreasing order are: polyvalent T(alpha), II(beta)/I(beta) and E(beta)>tri-antennary II(beta)>>monomeric T(alpha), T(beta), I and II>Gal>>GalNAc (weak). As one of type 2 RIP lectins, these recognition factors of the B chain are likely to be crucial for attachment and endocytosis. A comparison of the differential recognition factors and combining sites of APA with those of other lectins (Ricinus communis agglutinin, RCA(1) and ricin) is also illustrated.

Multivalent human blood group ABH and Lewis glycotopes are key recognition factors for a LFuc>Man binding lectin from phytopathogenic Ralstonia solanacearum.

Ralstonia solanacearum lectin (RSL), that might be involved in phytopathogenicity, has been defined as LFuc>>Man specific. However, the effects of polyvalency of glycotopes and mammalian structural units on binding have not been established. In this study, recognition factors of RSL were comprehensively examined with natural multivalent glycotopes and monomeric ligands using enzyme linked lectin-sorbent and inhibition assays. Among the glycans tested, RSL reacted strongly with multivalent blood group A(h) (GalNAcalpha1-3[Fucalpha1-2]Gal) and H (Fucalpha1-2Gal) active glycotopes, followed by B(h) (Galalpha1-3[Fucalpha1-2]Gal), Le(a) (Galbeta1-3[Fucalpha1-4]GlcNAc) and Le(b) (Fucalpha1-2Galbeta1-3[Fucalpha1-4]GlcNAc) active glycotopes. But weak or negligible binding was observed for blood group precursors having Galbeta1-3/4GlcNAcbeta1- (Ibeta/IIbeta) residues or Galbeta1-3GalNAcalpha1- (Talpha), GalNAcalpha1-Ser/Thr (Tn) bearing glycoproteins. These results indicate that the density and degree of exposure of multivalent ligands of alpha1-2 linked LFuc to Gal at the non-reducing end is the most critical factor for binding. An inhibition study with monomeric ligands revealed that the combining site of RSL should be of a groove type to fit trisaccharide binding with highest complementarity to blood group H trisaccharide (H(L); Fucalpha1-2Galbeta1-4Glc). The outstandingly broad RSL saccharide-binding profile might be related to the unusually wide spectrum of plants that suffer from R. solanacearum pathogenicity and provide ideas for protective antiadhesion strategies.

Expression of sialyl Lex, sialyl Lea, Lex and Ley glycotopes in secreted human ovarian cyst glycoproteins.

Human blood group A, B, H, Ii, Le(a) and Le(b) antigens and their determinants expressed on ovarian cyst glycoproteins have been studied for over five decades. However, little is known about sialyl Le(x) and sialyl Le(a) glycotopes, which play essential roles in normal immunity, inflammation, and cancer cell metastasis. Furthermore, Le(x) and Le(y) were classified as glycotopes of unknown genes. Identification of these Lewis epitopes was hampered by the lack of specific antibodies. In this study, the occurrence of sialyl Le(x), sialyl Le(a), Le(x) and Le(y) reactivities in cyst glycoproteins was characterized by enzyme-linked immunosorbent assays. The results indicated that most human ovarian cyst glycoproteins carried Le(x) (8/25) and/or Le(y) (17/25) glycotopes. The expression (epitopes) of the new genes described in previous reports are Le(x) and Le(y) glycotopes; the reactivities of sialyl Le(x) and sialyl Le(a) glycotopes in secreted cyst glycoproteins may be affected by the conditions of purification; the relationship between Le(y) and human blood group ABH was confirmed; recognition profiles of sialyl Le(x), sialyl Le(a), Le(x) and Le(y) present in the carbohydrate chains of water-soluble cyst glycoproteins were illustrated; possible attachments of glycotopes to the internal carbohydrate complex of cyst glycoproteins have been reconstructed; proposed biosynthetic pathways for the formation of sialyl Le(a), sialyl Le(x), Le(x), Le(y), ALe(y) and BLe(y) determinant structures on Type I and Type II core structures of human ovarian cyst glycoproteins are also included in this study.

Differential contributions of recognition factors of two plant lectins -Amaranthus caudatus lectin and Arachis hypogea agglutinin, reacting with Thomsen-Friedenreich disaccharide (Galbeta1-3GalNAcalpha1-Ser/Thr).

Previous reports on the carbohydrate specificities of Amaranthus caudatus lectin (ACL) and peanut agglutinin (PNA, Arachis hypogea) indicated that they share the same specificity for the Thomsen-Friedenreich (T(alpha), Galbeta1-3GalNAcalpha1-Ser/Thr) glycotope, but differ in monosaccharide binding--GalNAc>>Gal (inactive) for ACL; Gal>>GalNAc (weak) with respect to PNA. However, knowledge of the recognition factors of these lectins was based on studies with a small number monosaccharides and T-related oligosaccharides. In this study, a wider range of interacting factors of ACL and PNA toward known mammalian structural units, natural polyvalent glycotopes and glycans were examined by enzyme-linked lectinosorbent and inhibition assays. The results indicate that the main recognition factors of ACL, GalNAc was the only monosaccharide recognized by ACL as such, its polyvalent forms (poly GalNAcalpha1-Ser/Thr, Tn in asialo OSM) were not recognized much better. Human blood group precursor disaccharides Galbeta1-3/4GlcNAcbeta (I(beta)/II(beta)) were weak ligands, while their clusters (multiantennary II(beta)) and polyvalent forms were active. The major recognition factors of PNA were a combination of alpha or beta anomers of T disaccharide and their polyvalent complexes. Although I(beta)/II(beta) were weak haptens, their polyvalent forms played a significant role in binding. From the 50% molar inhibition profile, the shape of the ACL combining site appears to be a cavity type and most complementary to a disaccharide of Galbeta1-3GalNAc (T), while the PNA binding domain is proposed to be Galbeta1-3GalNAcalpha or beta1--as the major combining site with an adjoining subsite (partial cavity type) for a disaccharide, and most complementary to the linear tetrasaccharide, Galbeta1-3GalNAcbeta1-4Galbeta1-4Glc (T(beta)1-4L, asialo GM(1) sequence). These results should help us understand the differential contributions of polyvalent ligands, glycotopes and subtopes for the interaction with these lectins to binding, and make them useful tools to study glycosciences, glycomarkers and their biological functions.

Defining the carbohydrate specificities of Erythrina corallodendron lectin (ECorL) as polyvalent Galbeta1-4GlcNAc (II) > monomeric II > monomeric Gal and GalNAc.

BACKGROUND: Erythrina corallodendron lectin (ECorL) is one of the potent applied lectins. In previous studies, the carbohydrate specificities of this lectin were limited to monosaccharides, simple oligosaccharides and several clusters. However, the polyvalent factor has not been investigated. METHODS: The binding properties at the combining sites of ECorL were characterized by sensitive enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using our collection of ligands and polyvalent natural glycans with known glycotopes. RESULTS: Results of both binding and inhibition assays revealed a very high affinity between ECorL and Galbeta1-4GlcNAc (II)-containing glycoproteins. Among soluble natural glycans tested for inhibition, the high-density polyvalent II glycotopes, such as Streptococcus pneumoniae type 14 capsular polysaccharide which is composed of repeating poly-II residues, resulted in 2.4 x 10(4), 1.4 x 10(3) and 8.6 x 10(2)-fold higher affinities to ECorL than the monomeric Gal, linear II and tri-antennary II, respectively, at the non-reducing end in N-linked glycopeptides (Tri-II). The ECorL-glycan interaction was also strongly inhibited by most of the other high-density II-containing glycoproteins. Although GalNAc was as potent an inhibitor as Gal, its polyvalent structural units were poor inhibitors. CONCLUSIONS: [1] Galbeta1-4GlcNAc (II) and other Galbeta1 -related oligosaccharides are essential for binding. [2] Their polyvalent form in glycoproteins is the most important binding factor for ECorL, while II monomer and oligo-antennary II forms play only a limited role in binding. [3] Although GalNAc is more active than Gal for ECorL, its reactivity is not changed by polyvalent effects. This lectin may be used as a tool to study glycobiology in basic and medical sciences.

Differential affinities of Erythrina cristagalli lectin (ECL) toward monosaccharides and polyvalent mammalian structural units.

Previous studies on the carbohydrate specificities of Erythrina cristagalli lectin (ECL) were mainly limited to analyzing the binding of oligo-antennary Galbeta1-->4GlcNAc (II). In this report, a wider range of recognition factors of ECL toward known mammalian ligands and glycans were examined by enzyme-linked lectinosorbent and inhibition assays, using natural polyvalent glycotopes, and a glycan array assay. From the results, it is shown that GalNAc was an active ligand, but its polyvalent structural units, in contrast to those of Gal, were poor inhibitors. Among soluble natural glycans tested for 50% molecular mass inhibition, Streptococcus pneumoniae type 14 capsular polysaccharide of polyvalent II was the most potent inhibitor; it was 2.1 x 10(4), 3.9 x 10(3) and 2.4 x 10(3) more active than Gal, tri-antennary II and monomeric II, respectively. Most type II-containing glycoproteins were also potent inhibitors, indicating that special polyvalent II and Galbeta1-related structures play critically important roles in lectin binding. Mapping all information available, it can be concluded that: [a] Galbeta1-->4GlcNAc (II) and some Galbeta1-related oligosaccharides, rather than GalNAc-related oligosaccharides, are the core structures for lectin binding; [b] their polyvalent II forms within macromolecules are a potent recognition force for ECL, while II monomer and oligo-antennary II forms play only a limited role in binding; [c] the shape of the lectin binding domains may correspond to a cavity type with Galbeta1-->4GlcNAc as the core binding site with additional one to four sugars subsites, and is most complementary to a linear trisaccharide, Galbeta1-->4GlcNAcbeta1-->6Gal. These analyses should facilitate the understanding of the binding function of ECL.

Inhibition of corneal neovascularization with endostatin delivered by adeno-associated viral (AAV) vector in a mouse corneal injury model.

The use of a recombinant adeno-associated viral (rAAV) vector carrying endostatin gene as an anti-angiogenesis strategy to treat corneal neovascularization in a mouse model was evaluated. Subconjunctival injection of recombinant endostatin-AAV was used to examine the inhibition of corneal neovascularization induced by silver nitrate cauterization in mice. The results showed that gene expression in corneal tissue was observed as early as 4 days after gene transfer and stably lasted for over 8 months with minimal immune reaction. Subconjunctival injection of a high-titer rAAV-endostatin successfully inhibited neovascularization. Immunohistchemistry staining of CD 31 and endostatin showed that the treatment significantly inhibits angiogenesis in cornea. We concluded that the rAAV was capable of directly delivering genes to the ocular surface epithelium by way of subconjunctival injection and was able to deliver sustained, high levels of gene expression in vivo to inhibit angiogenesis.

Recognition profile of Morus nigra agglutinin (Morniga G) expressed by monomeric ligands, simple clusters and mammalian polyvalent glycotopes.

The carbohydrate binding properties of a novel member of the subfamily of galactose-specific jacalin-related lectin isolated from the bark of black mulberry (Morus nigra) (Morniga G) was studied in detail by enzyme-linked lectinosorbent and inhibition assays using panels of monomeric saccharides, mammalian polyvalent glycotopes and polysaccharides. Among the natural glycans tested for lectin binding, Morniga G reacted best with glycoproteins (gps) presenting a high density of tumor-associated carbohydrate antigens Tn (GalNAcalpha1-Ser/Thr) and Talpha (Galbeta1-3GalNAcalpha1-). Their reactivities, on a nanogram basis, were up to 72.5, 3.9x10(3), 6.0x10(3), 8.8x10(3) and 2.9x10(4) times higher than that of Tn-containing glycopeptides (M.W.<3000 Da), monomeric T, Tn, GalNAc and Gal, respectively. It also reacted well with many multi-antennary N-glycans with II (Galbeta1-4GlcNAc) termini, ABH histo-blood group antigens and their precursors containing high densities of I/II and T/Tn glycotopes, and sialylated T/Tn. Among the mono-, di- and oligosaccharides tested, Thomsen-Friedenreich (T) disaccharide with aromatic aglycon [Galbeta1-3GalNAcalpha1-benzyl (Talpha1-benzyl)] and Tn glycopeptides were the best inhibitors. Molecular modeling and docking studies indicated the occurrence of a primary GalNAcalpha1- and Galbeta1-3GalNAc glycotope-binding site in Morniga G. Using a recently proposed system [Wu, A.M., 2003. Carbohydrate structural units in glycoproteins and polysaccharides as important ligands for Gal and GalNAc reactive lectins. J. Biomed. Sci. 10, 676-688], the binding properties of the combining sites of Morniga G can be defined as follows: (i) the monosaccharide specificity is GalNAc/Gal>>Man/Glc, GlcNAc and lFuc; (ii) the mammalian glycotope specificity is Talpha1-benzyl>T>Tn>GalNAcbeta1-3Gal (P), while B/E (Galalpha1-3/4Gal), I/II (Galbeta1-3/4GlcNAc), S (GalNAcbeta1-4Gal), F/A (GalNAcalpha1-3GalNAc/Gal) and L (Galbeta1-4Glc) are inactive; (iii) the most active ligand is T/Tn; (iv) simple clustered Tn or triantennary N-glycans with II termini (Tri-II) have limited impact; (v) high-density polyvalent glycotopes play a prominent role for enhancing Morniga G reactivity. These results provide evidence for the binding of this lectin to dense cell surface T/Tn glycoconjugates and facilitate future usage of this lectin in biotechnological and medical applications.

Mitogen-activated protein kinase (MAPK) signalling pathways in HepG2 cells infected with a virulent strain of Klebsiella pneumoniae.

Klebsiella pneumoniae (KP), an enterobacterium, usually causes urinary tract infection or pneumonia; however, it has caused severe liver abscess in diabetic patients in recent years. How this emerging virulent KP strain causes liver abscess is not known. This study investigates signalling pathways in HepG2 cells infected by virulent KP. Cells were infected with bacteria for various durations and harvested to screen for signalling molecules by Western blotting. Our results showed that phosphorylated mitogen-activated protein kinase (MAPK) kinase (MEK) 1/2, p44/p42 MAPK and p90 ribosomal S6 kinase (p90RSK) were observed and this pathway was inhibited by MEK1/2 inhibitors U0126 and PD98059. Phosphorylation of MEK3/6, p38 kinase and ATF-2 was also observed and this pathway was inhibited by p38 kinase inhibitors SB203850 and SB202190. Toll-like receptor (TLR) 2 and 4 expressions were increased and maximized 2-4 h post infection. The JNK pathway, Elk, MAPKAPK-2 and HSP27 were not activated. These results suggest that KP infections induce signal transduction through TLR2 and TLR4 and activate two downstream MAP kinase pathways, MEK1/2-p44/p42 MAPK-p90RSK and MEK3/6-p38 kinase-ATF-2, but not the JNK pathway in HepG2 cells. The infected HepG2 eventually showed apoptosis and died.

Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: probing its fine specificity and the effect of naturally clustered ligand presentation.

Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galbeta1-3/4GlcNAc (I/II) and multiantennary N-glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 x 10(2), 3.2 x 10(2), 2.5 x 10(2), and 1.7 x 10(4) times higher than monomeric Galbeta1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galalpha1-3Gal)- and A (GalNAcalpha1-3Gal)-containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcalpha1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galalpha1-3Gal beta1-4GlcNAc), B-active IIbeta1-3L (Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc), and Tri-II were the best. It is concluded that (1) Galbeta1-3/4GlcNAc and other Galbeta1-related oligosaccharides with alpha1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted beta-galactoside, especially with alpha-anomeric extension at the non-reducing end (e.g., human blood group B-active II and B-active IIbeta1-3L); (3) the preference within beta-anomeric positioning is Galbeta1-4 > or = Galbeta1-3 > Galbeta1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/growth-regulatory effectors with potential for medical applications.

Carbohydrate specificity of an insecticidal lectin isolated from the leaves of Glechoma hederacea (ground ivy) towards mammalian glycoconjugates.

Preliminary studies indicated that the potent insecticidal lectin, Gleheda, from the leaves of Glechoma hederacea (ground ivy) preferentially agglutinates human erythrocytes carrying the Tn (GalNAcalpha1-Ser/Thr) antigen. However, no details have been reported yet with respect to the fine specificity of the lectin. To corroborate the molecular basis of the insecticidal activity and physiological function of Gleheda, it is necessary to identify the recognition factors that are involved in the Gleheda-glycotope interaction. In the present study, the requirement of high-density multivalent carbohydrate structural units for Gleheda binding and a fine-affinity profile were evaluated using ELLSA (enzyme-linked lectinosorbent assay) with our extended glycan/ligand collections, a glycan array and molecular modelling. From the results, we concluded that a high-density of exposed multivalent Tn-containing glycoproteins (natural armadillo and asialo ovine salivary glycoproteins) were the most potent factors for Gleheda binding. They were, on a nanogram basis, 6.5x10(5), 1.5x10(4) and 3.1x10(3) times more active than univalent Gal (galactose), GalNAc (N-acetylgalactosamine) and Tn respectively. Among mono- and oligo-saccharides examined, simple clustered Tn (molecular mass <3000 Da) from ovine salivary glycoprotein was the best, being 37.5 and 1.7x10(3) times better than GalNAc and Gal respectively. GalNAc glycosides were significantly more active than Gal glycosides, indicating that the N-acetamido group at C-2 plays an important role in Gleheda binding. The results of glycan array support the conclusions drawn with respect to the specificity of Gleheda based on the ELLSA assays. These findings combined with the results of the molecular modelling and docking indicate the occurrence of a primary GalNAcalpha1-binding site in the Gleheda monomer. However, the extraordinary binding feature of Gleheda for glycoproteins demonstrates the importance of affinity enhancement by high-density multivalent glycotopes in the ligand-lectin interactions in biological processes.

Carbohydrate recognition factors of the lectin domains present in the Ricinus communis toxic protein (ricin).

Ricin (RCA60) is a potent cytotoxic protein with lectin domains, contained in the seeds of the castor bean Ricinus communis. It is a potential biohazard. To corroborate the biological properties of ricin, it is essential to understand the recognition factors involved in the ricin-glycotope interaction. In previous reports, knowledge of the binding properties of ricin was limited to oligosugars and glycopeptides with different specificities. Here, recognition factors of the lectin domains in ricin were examined by enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using mammalian Gal/GalNAc structural units and corresponding polyvalent forms. Except for blood group GalNAcalpha1-3Gal (A) active and Forssman (GalNAcalpha1-3GalNAc, F) disaccharides, ricin has a broad range of affinity for mammalian disaccharide structural units-Galbeta1-4Glcbeta1-(Lbeta), Galbeta1-4GlcNAc (II), Galbeta1-3GlcNAc (I), Galbeta1-3GalNAcalpha1-(Talpha), Galbeta1-3GalNAcbeta1-(Tbeta), Galalpha1-3Gal (B), Galalpha1-4Gal (E), GalNAcbeta1-3Gal (P), GalNAcalpha1-Ser/Thr (Tn) and GalNAcbeta1-4Gal (S). Among the polyvalent glycotopes tested, ricin reacted best with type II-containing glycoproteins (gps). It also reacted well with several T (Thomsen-Friedenreich), tumor-associated Tn and blood group Sd. (a+)-containing gps. Except for bird nest and Tamm-Horsfall gps (THGP), this lectin reacted weakly or not at all with ABH-blood type and sialylated gps. From the present and previous results, it can be concluded that: (i) the combining sites of these lectin domains should be a shallow-groove type, recognizing Galbeta1-4Glcbeta1- and Galbeta1-3(4)GlcNAcbeta- as the major binding site; (ii) its size may be as large as a tetrasaccharide and most complementary to lacto-N-tetraose (Galbeta1-3GlcNAc beta1-3Galbeta1-4Glc) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc); (iii) the polyvalency of glycotopes, in general, enhances binding; (iv) a hydrophobic interaction in the vicinity of the binding site for sugar accommodation, increases the affinity for Galbeta-. This study should assist in understanding the glyco-recognition factors involved in carbohydrate-toxin interactions in biological processes. The effect of the polyvalent P/S glycotopes on ricin binding should be examined. However, this is hampered by the lack of availability of suitable reagents.

Recognition factors of Ricinus communis agglutinin 1 (RCA(1)).

Ricinus communis agglutinin (RCA1) is one of the most important applied lectins that has been widely used as a tool to study cell surfaces and to purify glycans. Although the carbohydrate specificity of RCA1 has been described, the information obtained was mainly focused on inhibition of simple Galbeta1-related oligosaccharides and simple clusters. Here, all possible recognition factors of RCA1 of glycan binding were examined by enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using known mammalian Gal/GalNAc carbohydrate structural units and natural polyvalent glycans. Among the glycoproteins (gps) tested and expressed as 50% nanogram inhibition, the high-density polyvalent Galbeta1-4GlcNAc (II) glycotopes occurring in natural gps, such as Pneumococcus type 14 capsular polysaccharide which is composed of repeating poly II residues, resulted in 9.0 x 10(4), 1.5 x 10(5), 2.3 x 10(4) and 2.1 x 10(4)-fold higher affinities to RCA1 than the monomeric Gal, linear I/II and Tri-antennary-II (Tri-II). Of the ligands tested and expressed as nanomoles of 50% inhibition, Tri-II was the best, being about 2, 4, 25.6 and 33.3 times better inhibitor than Di-II, II, I (Galbeta1-3GlcNAc) and Gal, respectively. From the results of this study, it is concluded that: (a) Galbeta1-4GlcNAc and other Galbeta1-related oligosaccharides are essential for lectin binding and their polyvalent form in macromolecules should be the most important recognition factor for RCA1; (b) the combining site of RCA1 may be a groove type, recognizing Galbeta1-4GlcNAc (II) as the major binding site; (c) its combining size may be large enough to accommodate a tetrasaccharide of beta-anomeric Gal at the non-reducing end and most complementary to human blood group I Ma active trisaccharide (Galbeta1-4GlcNAcbeta1-6Gal) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc); (d) RCA1 has a preference for the beta-anomer of Gal oligosaccharides with a Galbeta1-4 linkage > Galbeta1-6 > or = Galbeta1-3; (e) configuration of carbon-2, -3 -4 and -6 in Gal are essential for binding; (f) hydrophobic interaction in the vicinity of the binding site useful for sugar accommodation increases affinity. These results should be helpful for understanding the functional role of RCA1 and for characterizing glycotopes of mammalian complex carbohydrates.

Lectinochemical studies on the binding properties of a toxic lectin (ricin) isolated from the seeds of Ricinus communis.

BACKGROUND: Ricin (RCA2 or RCA60) is a highly toxic heterodimeric protein found in the seeds of the castor plant Ricinus communis. It is a potential biohazard. In the present study, the fine specificity of ricin was defined. METHODS: The combining site of ricin was characterized by quantitative precipitin (QPA) and precipitin inhibition assays (QPIA). RESULTS: Of 31 glycoproteins and pneumococcus type XIV capsular polysaccharide tested, only twelve of them precipitated over 50% of the toxin N added, reflecting poor precipitability of the lectin with the compounds tested. This can be explained by only a single chain (B chain of the molecules) participating in binding. The blood group active glycoproteins after mild acid hydrolysis or Smith degradation, as well as sialic-acid containing glycoproteins after removal of sialic acid, in general, had substantially increased activity. Of the monosaccharides tested for inhibition of precipitation of ricin, p-nitrophenyl betaGal was the best; this compound was 1.3-fold better than its alpha-anomer. While methyl betaGal was twice as active as its alpha anomer, Gal and blood group B active disaccharides (Galalpha1-3Gal) were 2.5 times more active than GalNAc. Among the oligosaccharides tested, Galbeta1-3GalNAc (T) Gal beta1-3/4GlcNAc (I/II), Galbeta1-4Glc (L) and human blood group I Ma trisaccharide (Galbeta1-4GlcNAcbeta1-6Gal) were about equally active and the best inhibitors. They were about 2.0 and 2.4 more active than Galalpha1-4Gal (E) sequence and B determinant, respectively. CONCLUSION: From the present results, it is concluded that: (a) this toxin has a broad range of affinity for the beta-anomer of Gal; (b) its combining site is probably of a shallow groove type and as large as a trisaccharide; (c) Galbeta--is the major combining site of the lectin; and (d) hydrophobic interaction gives a significant contribution for binding. This information should facilitate future usage of this lectin in glycobiological research and medical applications.

Dexamethasone enhances upregulation of nerve growth factor mRNA expression in ischemic rat brain.

Nerve growth factor (NGF) is well-established as a trophic factor that plays a crucial role in neuroregeneration and plasticity after brain insults. Dexamethasone (DEX), a powerful glucocorticoid steroid, has long been used in the clinical management of neurological disorders. We examined the relationship between NGF and DEX after an ischemic insult to the brain. In situ hybridization was used to measure NGF mRNA expression in the rat hippocampus after 20 min of transient forebrain ischemia. Immunostaining for NGF protein was performed using the avidin-biotin peroxidase method. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was also used to study the astrocyte reaction in the hippocampal CA1 area. Ischemic brain from rats not treated with DEX had a 2 and 3 fold increase in NGF mRNA compared to sham-operated rats at 4 and 6 h after ischemia, respectively. The NGF mRNA expression returned to basal levels 12 h to 7 days post-ischemia. Treatment with DEX potentiated the ischemia-induced increase of NGF mRNA to 4 times that of sham-operated rats at 6 h following reperfusion and NGF protein expression was similarly elevated. Additionally, the number of GFAP positive astrocytes in the CA1 region in the ischemic rats was markedly increased. These data suggest that DEX may play a role in modulating NGF mRNA expression in the hippocampal neuronal response to brain ischemia.