Synthesis of Galβ1-4GlcNAcβ- and Galβ1-3GalNAcα-O-L-serine Derivatives employing glycosidases

A new approach for the highly specific preparation of L-serine conjugates of lactosamine and Gal1-3GalNAc is described. Thus, the L-serine derivative of lactosamine Gal1-4GlcNAc-O-(N-Z)-Ser-OEt, was obtained from lactose, employing GlcNAc-O-(N-Z)-Ser-OEt as acceptor and a yeast -galactosidase as catalyst Galp 1-3GalNAc-O-(N-Alloc)-Ser-OMe was obtained from lactose, employing GalNAc-O-(N-Alloc)-Ser-OMe as acceptor and -galactosidase from bovine testes as catalyst.     

Concanavalin A-stimulated expression of gangliosides with GalNAcβ1-4(NeuAcα2-3)Galβ structure in murine thymocytes

We analysed the glycolipids of mouse thymocytes before and after Concanavalin A (Con A) or recombinant interleukin-2 (rIL-2) stimulation by TLC-immunostaining with carbohydrate-specific antiglycolipid antibodies. The thymocytes were cultured in serum-free medium in the presence of 500 ng ml^–1 Con A, 10 U ml^–1 rIL-2 or Con A plus rIL-2 for 6, 12, 24, 48, and 72 h, and were found to start proliferating 24 h after cultivation in the presence of Con A or Con A plus rIL-2, the maximum levels being reached at 72 h and 48 h, respectively, in a thymidine uptake experiment. The concentrations of II³Neu-Gg₄Cer, Gg₄Cer and IV³GalNAc-Gb₄Cer after 48 h Con A stimulation were found to be at almost the original levels. Conversely, II³Neu-Gg₃Cer, which was not detected in the thymocytes at the start, began to appear after 48 h stimulation with Con A and Con A plus rIL-2, and IV³Neu-Gg₅Cer in the cells 48 h after stimulation with Con A and Con A plus rIL-2 has increased to 41 and 44 times higher than in the original cells, respectively, as judged on TLC-immunostaining with monoclonal antibody YHD-06, which detects the GalNAc1-4(NeuAc or NeuGc2-3)Gal-structure. These results indicate that the increased synthesis of both gangliosides, in other words, the activation ofN-acetylgalactosaminyltransferase, is associated with the mitogen-induced proliferation.N-Acetylneuraminic acid was the sole sialic acid in II³Neu-Gg₃Cer which newly appeared in the cells on stimulation, whereas the sialic acid of IV³Neu-Gg₅Cer was a mixture ofN-acetyl- andN-glycoloylneuraminic acids. This result may suggest that the substrates for the two differentN-acetylgalactosaminyltransferases must be different. This GalNAc1-4(NeuAc or NeuGc2-3)Gal-structure was also detected on the surface of the Con A or Con A plus rIL-2 stimulated mouse thymocytes on flow cytometric analysis of cells indirectly stained with monoclonal antibody YHD-06. Abbreviations: carbohydrate and glycolipid nomenclature and abbreviations follow the IUPAC-IUB recommendations or the nomenclature system of Svennerholm L. (1963)J Neurochem 10:613–23.     

ProcessingO-glycan core 1, Galβ1-3GalNAcα-R. Specificities of core 2, UDP-GlcNAc: Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyltransferase and CMP-sialic acid:Galβ1-3GalNAc-R α3-sialyltransferase

To elucidate control mechanisms ofO-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Gal1-3GalNAc-R(GlcNAc to GalNAc) 6-N-acetylglucosaminyl-transferase (EC 2.4.1.102; core 2 6-GlcNAc-T) and CMP-sialic acid: Gal1-3GalNAc-R 3-sialyltransferase (EC 2.4.99.4; 3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 6-GlcNAc-T activity; core 2 6-GlcNAc-T from mucin secreting tissue (named core 2 6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAc1-6(GlcNAc1-3)GalNAc-R] and blood group I [GlcNAc1-6(GlcNAc1-3)Gal-R] branches; core 2 6-GlcNAc-T in leukemic cells (named core 2 -GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls ofN-acetylgalactosamine and the 6-hydroxyl of galactose of the Gal1-3GalNAc-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. 3-sialytransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Gal1-3GalNAc-Bn. Gal1-3(6-deoxy)GalNAc-Bn and 3-deoxy-Gal1-3GalNAc-Bn competitively inhibited core 2 6-GlcNAc-T and 3-sialyltransferase activities, respectively.Abbreviations AFGP antifreeze glycoprotein - AML acute myeloid leukemia - Bn benzyl - CML chronic myelogenous leukemia - Fuc l-fucose - Gal, G d-galactose - GalNAc, GA N-acetyl-d-galactosamine - GlcNAc, Gn N-acetyl-d-glucosamine - HC human colonic homogenate - HO hen oviduct microsomes - HPLC high performance liquid chromatography - mco 8-methoxycarbonyl-octy - Me methyl - MES 2-(N-morpholino)ethanesulfonate - MK mouse kidney homogenate - onp o-nitrophenyl - PG pig gastric mucosal microsomes - pnp p-nitrophenyl - RC rat colonic mucosal microsomes - SA sialic acid - T transferase Enzymes: UDP-GlcNAc:Gal1-3GalNAc-R (GlcNAc to GalNAc) 6-N-acetylglucosaminyltransferase,O-glycan core 2 6-GlcNAc-transferase, EC 2.4.1.102; CMP-sialic acid: Gal1-3GalNAc-R 3-sialyltransferase,O-glycan 3-sialic acid-transferase, EC 2.4.99.4.     

Presence of natural anti-Galα1-4GalNAcβ1-3Gal (anti-NOR) antibodies in animal sera

Rare polyagglutinable NOR erythrocytes contain unusual globoside extention products terminating with a Galα1-4GalNAcβ1-3Gal- unit. This trisaccharide epitope is recognized by recently characterized antibodies naturally occurring in most human sera (Duk et al., Glycobiology, 15, 109, 2005). These antibodies represent two major types of fine specificity. All these antibodies are most strongly inhibited by Galα1-4GalNAcβ1-3Gal (NOR-tri), and weakly by Galα1-4Gal. However, the type 1 antibodies are strongly inhibited by Galα1-4Galβ1-3Gal-R and weakly by Galα1-4GalNAc, while the type 2 antibodies show the opposite reactivities with these two oligosaccharides. Similar antibodies have now been found in horse, rabbit and pig sera. The antibodies were purified from animal sera by affinity chromatography on Galα1-4GalNAcβ1-3Gal-human serum albumin(HSA)-Sepharose 4B conjugate. The specificity of the antibodies was determined by binding to ELISA plates coated with several α-galactosylated oligosaccharide-polyacrylamide (PAA) or -HSA conjugates and by inhibition with synthetic oligosaccharides. The purified antibodies bound specifically to conjugates containing NOR-tri. The inhibition of binding showed that the animal sera also contain two types of anti-NOR antibodies: type 2 was found in the horse serum, and a mixture of both types was present in rabbit and pig serum. These results indicate that anti-NOR, a new and distinct kind of anti-αGal antibody, are present in animal sera and show similar specificties and diversity as their counterparts found in human sera.     

The α-Gal epitope (Galα1-3Galβ1-4GlcNAc-R) in xenotransplantation

Many patients with failing organs (e.g., heart, liver or kidneys), do not receive the needed organ because of an insufficient number of organ donors. Pig xenografts have been considered as an alternative source of organs for transplantation. The major obstacle currently known to prevent pig to human xenotransplantation is the interaction between the human natural anti-Gal antibody and the alpha-gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R), abundantly expressed on pig cells. This short review describes the characteristics of anti-Gal and of the alpha-gal epitope, their role in inducing xenograft rejection and some experimental approaches for preventing this rejection.     

Different reactivity of two Brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAcα2-3Galβ1-3GalNAc α(2–6)sialyltransferase

We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited byN-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialytransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl₂ did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group, is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.Abbreviations p-CMB p-chloromercuribenzoic acid - CPDS 6,6-dithiodinicotinic acid carboxypyridine disulfide - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - DTT dithiothreitol - Mes 2-(N-morpholino)ethane sulfonic acid - NeuAc N-acetylneuraminic acid     

A monoclonal antibody specific for non-reducing terminal Galα1-4Gal residues

A mouse monoclonal antibody (87.5) against Gal1-4Gal has been obtained after immunization with the disaccharide glycosidically coupled to a protein. The specificity was determined by studying its binding to a number of glycoconjugates and oligosaccharides.The antibody which was found to be highly specific for terminal Gal1-4Gal residues is a powerful tool for the detection of this structure in glycoproteins and glycolipids by immunochemicalin vitro methods. It is also useful forin vitro quantification of the free disaccharide.A thin layer chromatographic overlay assay using glycolipids and an immunoperoxidase technique is also described. The antibody 87.5 is used in this assay to identify human uroepithelium glycolipids with terminal Gal1-4Gal residues.Abbreviations Lactosylceramide Gal1-4GlcCer - globotriaosylceramide GbOse₃-ceramide, Gal1-4Gal1-4GlcCer - globotetraosylceramide globoside, GbOse₄-ceramide, GalNAc1-3Gal1-4Gal1-4GlcCer     

Unique disialosyl gangliosides from salmon kidney: Characterization of V3αFuc, IV3βGalNAc, II3(αNeuAc)2-Gg4Cer and its analogue with 4-O-acetyl-N-acetylneuraminic acid

Four unidentified acidic glycolipids (X3-X6) were isolated from the kidney of the Pacific salmon on an anion exchange column and by high performance liquid chromatography using a silica bead (Iatrobeads) column. Based on methylation analysis, chemical and enzymatic degradation, proton nuclear magnetic resonance spectroscopy and mass spectrometry, the glycon structure of X5 and X6 was identified as a unique disialosyl fucosyl-N-acetylgalactosaminyl ganglio-N-tetraose: Fucα3GalNAcβ3Galβ3GalNAcβ4[NeuAcα8NeuAcα3] Galβ4Glcβ1Cer. NMR showed that X3 and X4 were analogues of X5 and X6 and contained O-acetyl groups on C4 of the outer N-acetylneuraminic acid, first disialosyl gangliosides containing 4-O-acetyl-N-acetylneuraminic acid. The ceramides of X3 and X5 contained predominantly C24: 1, and X4 and X6 contained saturated fatty acids (C14: 0, C16: 0 and C18: 0), whereas the long chain base was exclusively sphingenine. The concentrations of X3 and X4 were 0.13 and 0.16 nmol/g of kidney respectively and those of X5 and X6, were 0.07 nmol/g each.     

Differential Binding to Glycotopes Among the Layers of Three Mammalian Retinal Neurons by Man-Containing N-linked Glycan, Tα (Galβ1–3GalNAcα1-), Tn (GalNAcα1-Ser/Thr) and Iβ/IIβ (Galβ1–3/4GlcNAcβ-) Reactive Lectins

Carbohydrate structures between retinal neurons and retinal pigment epithelium (RPE) play an important role in maintaining the integrity of retinal adhesion to underlying RPE, and in retinal detachment pathogenesis. Since relevant knowledge is still in the primary stage, glycotopes on the adult retina of mongrel canines (dog), micropigs and Sprague-Dawley rats were examined by lectino-histochemistry, using a panel of 16 different lectins. Paraffin sections of eyes were stained with biotinylated lectins, and visualized by streptavidin-peroxidase and diaminobenzidine staining. Mapping the affinity profiles, it is concluded that: (i) all sections of the retina reacted well with Morniga M, suggesting that N-linked glycans are present in all layers of the retina; (ii) no detectable human blood group ABH active glycotopes were found among retinal layers; (iii) outer and inner segments contained glycoconjugates rich in ligands reacting with T _α (Galβ1–3GalNAcα1-Ser/Thr) and Tn (GalNAcα1-Ser/Thr) specific lectins; (iv) cone cells of retina specifically bound peanut agglutinin (PNA), which recognizes T _α residues and could be used as a specific marker for these photoreceptors; (v) the retinas of rat, dog and pig, had a similar binding profile but with different intensity; (vi) each retinal layer had its own binding characteristic. This information may provide useful background knowledge for normal retinal physiology and miscellaneous retinal diseases, including retinal detachment (RD) and age-related macular degeneration (ARMD).     

Synthesis of 1α-[2-H]Hydroxyvitamin D3

1α-[2-³H]Hydroxyvitamin D₃ was synthesized chemically. The preparation was radiochemically pure and had a high enough specific activity (4.2 Ci/mmol) to permit experiments using 62.5 pmol/rat. This preparation had as much effect as synthetic 1α-hydroxyvitamin D₃ in increasing the serum Ca level of vitamin D-deficient rats.     

Enzymic glycosphingolipid synthesis on polymer supports. III. Synthesis of GM3, its analog [NeuNAcα(2-3)Galβ(1-4)Glcβ(1-3)Cer] and their lyso-derivatives

Two water-soluble polymers, carrying 0.24 meq g–1 of lactosyl-(1-1)-sphingosine (7) and 0.13 meq g–1 of lactosyl-(1-3)-sphingosine (8) were prepared. The polymers served as acceptors in the -(2-3)-sialyltransferase reaction (up to 55.3 and 38.5% transfer yields, respectively). Subsequent photolysis, released compounds 11 (lyso-GM3) and 12 (lyso-GM3 analog), respectively; acylation and chromatography afforded (5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonic acid)-(2-3)--D-galactopyranosyl-(1-4)--D-glucopyranosyl-(1-1)-(2S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (13, GM3) and (5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonic acid)-(2-3)--D-galactopyranosyl-(1-4)--D-glucopyranosyl-(1-3)-(2S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (14, GM3 analogue), respectively, thus presenting a route to glycosphingolipids possessing the unusual glycosyl-(1-3)-spingosine linkage.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

Probe design and synthesis of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of N-glycan

Synthesis and clusterization of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of the N-glycan, as the molecular probes for their biological evaluation, are reported. Key step is the quantitative and the completely α-selective sialylation of the C5-azide N-phenyltrifluoroacetimidate with the disaccharide acceptor, Galβ(1→3)GlcNTroc. Clusterization of the 16 molecules of trisaccharide motif was also achieved by the ‘self-activating click reaction’. These probes could efficiently be labeled by biotin and/or other fluorescence- or radioactive reporter groups through either cross metathesis, acylation, Cu(I)-mediated Huisgen [2+3]-cycloaddition, or the azaelectrocyclization to utilize the various biological techniques.     

Vaccination-induced IgG response to Galα1-3GalNAc glycan epitopes in lambs protected against Haemonchus contortus challenge infection

Lambs vaccinated with Haemonchus contortus excretory/secretory (ES) glycoproteins in combination with the adjuvant Alhydrogel are protected against H. contortus challenge infection. Using glycan micro-array analysis we showed that serum from such vaccinated lambs contains IgG antibodies that recognise the glycan antigen Galα1-3GalNAc-R and GalNAcβ1-4(Fucα1-3)GlcNAc-R. Our studies revealed that H. contortus glycoproteins contain Galα1-3Gal-R as well as significant levels of Galα1-3GalNAc-R, which has not been previously reported. Extracts from H. contortus adult worms contain a galactosyltransferase acting on glycan substrates with a terminal GalNAc, indicating that the worms possess the enzymatic potential to synthesise terminal Gal-GalNAc moieties. These data illustrate that glycan micro-arrays constitute a promising technology for fast and specific analysis of serum anti-glycan antibodies in vaccination studies. In addition, this approach facilitates the discovery of novel, antigenic parasite glycan antigens that may have potential for developing glycoconjugate vaccines or utilization in diagnostics.     

Cytochemical localization of GalNAc and GalNAcβ1,4Galβ1,4 disaccharide in mouse zona pellucida

Carbohydrate residues contained in the zona pellucida play a key role in the process of sperm-egg interaction. In vitro fertilization experiments have shown that a specific monoclonal antibody against GalNAcş,4Galş,4 disaccharide inhibits fertilization in mice. In the present study, the ultrastructural cytochemical localization of GalNAc residues and the GalNAcş,4Galş,4 disaccharide was carried out in ovarian and postovulatory oocytes by using lectin-gold cytochemistry and immunocytochemistry. Plant lectins SBA and DBA showed an affinity for the entire zona pellucida matrix of ovarian oocytes throughout the follicular maturation; however, immunoreactivity for GalNAcş,4Galş,4 disaccharide was not detected in ovarian oocytes at the earliest stages of follicular development but was found to be associated with the inner region of the zona matrix at the trilaminar primary follicle stage. The Golgi apparatus, vesicular aggregates, and cortical granules of the oocyte were intensely labeled by SBA and DBA throughout follicular development. Immunoreactivity to GalNAcş,4Galş,4 disaccharide was first observed in the Golgi apparatus and vesicular aggregates in trilaminar primary follicles. No immunoreactivity was observed in the cortical granules. In postovulatory oocytes, results were similar to those observed in ovarian oocytes. Our results thus suggest that (1) GalNAcş,4Galş,4 disaccharide residues are present only in the inner region of the zona pellucida and, therefore, might be involved in sperm penetration through the zona pellucida, (2) the inner and outer regions of the zona pellucida contain different oligosaccharide chains, (3) the vesicular aggregates detected in the oocyte could represent an intermediate step in the secretory pathway of zona pellucida glycoproteins and might be involved in the formation of cortical granules.     

Monoclonal antibodies that recognize the trisaccharide epitope Galα1-3Galβ1-4GlcNAc present on Ehrlich tumor cell membrane glycoproteins

Monoclonal antibodies were prepared against the trisaccharide Gal1-3Gal1-4GlcNAc, a sequence which occurs on the surface of Ehrlich ascites tumor cells as well as in thyroglobulin, laminin and a variety of other proteins. This was accomplished by immunizing BALB/c mice with the fraction of Ehrlich cell membrane glycoproteins obtained by affinity chromatography on aGriffonia simplicifolia I (GS I) column which selectively binds -d-galactosyl-terminated structures. Detection of Gal1-3Gal1-4GlcNAc-specific antibodies was accomplished by employing glycoproteins containing the trisaccharide sequence; fusion with spleen cells from an immunized mouse was accomplished in the presence of polyethylene glycol (PEG1500). An enzyme-linked immunosorbent assay (ELISA) system was used to identify two clones (2.10G and 6.8E), which recognized the desired trisaccharide conjugate. These clones also recognized a thyroglobulin fraction isolated by GS I affinity chromatography and murine laminin, both of which possess the Gal1-3Gal1-4GlcNAc sequence. Inhibition of antibody-trisaccharide reactivity, examined employing an ELISA assay, revealed that two trisaccharides, Gal1-3Gal1-4GlcNAc/Glc, were the best inhibitory haptens; Gal1-4GlcNAc (LacNAc), Gal1-3Gal and Gal1-4Glc (lactose) were poor inhibitors. Indirect immunofluorescence staining of unfixed Ehrlich cells using the monoclonal antibody at 4° C revealed fluorescence over the entire cell surface. Indirect immunogold labeling of semithin and ultrathin sections of aldehyde fixed and Lowicryl K4M-embedded Ehrlich cells resulted in specific labeling of the cell surface and internal structure. Immunoblot analysis revealed that removal of the -galactosyl residues of laminin by -galactosidase abolished reactivity with the monoclonal antibodies. The availability of this antibody, which belongs to the IgM family of immunoglobulins, now makes possible the detection of this sugar sequence on cells and tissue sections, as well as on glycoproteins in solution.     

Synthesis and radiopharmacological investigation of 3-[4′-[18F]fluorobenzylidene]indolin-2-one as possible tyrosine kinase inhibitor

The radiosynthesis and radiopharmacological evaluation of 3-[4′-[¹⁸F]fluorobenzylidene]indolin-2-one, a derivative of tyrosine kinase inhibitor SU5416, is described. The radiosynthesis was accomplished by Knoevenagel condensation of 4-[¹⁸F]fluorobenzaldehyde with oxindole in a remotely controlled synthesis module. The reaction conditions were optimized through screening the influence of different bases on the radiochemical yield. The radiotracer was obtained after a two-step labelling procedure in 4% decay-corrected radiochemical yield at a specific activity of 48–61 GBq/μmol within 90 min. The radiochemical purity after semi-preparative HPLC purification exceeded 98%.The biodistribution was studied in Wistar rats. After distribution the radiotracer was rapidly accumulated in the adrenals, liver and kidneys, however, it was cleared from these and the most other organs. Only the adipose tissue remained the activity over 60 min. Unexpected high transient uptake was observed in the brain, pancreas, heart and lung. The fast clearance of 3-[4′-[¹⁸F]fluorobenzylidene]indolin-2-one was caused by excretion, approximately one half each was renal and biliary excreted and the other part cleared by metabolic processes. In arterial blood plasma two more polar metabolites were found by radio-HPLC. After 20 min post-injection, only 12% of intact radiotracer has been detected. Consequently, in small animal PET studies with FaDu tumour bearing mice no specific uptake in the tumours could be observed.     

Occurrence of the human tumor-specific antigen structure Galβ1-3GalNAcα- (Thomsen-Friedenreich) and related structures on gut bacteria: prevalence, immunochemical analysis and structural confirmation

The Thomsen-Friedenreich antigen (TF; CD176, Galβ1-3GalNAcα-) is a tumor-specific carbohydrate antigen and a promising therapeutic target. Antibodies that react with this antigen are frequently found in the sera of healthy adults and are assumed to play a role in cancer immunosurveillance. In this study, we examined the occurrence of α-anomeric TF (TFα) on a large variety of gastrointestinal bacteria using a novel panel of well-characterized monoclonal antibodies. Reactivity with at least one anti-TF antibody was found in 13% (16 of 122) of strains analyzed. A more in-depth analysis, using monoclonal antibodies specific for α- and β-anomeric TF in combination with periodate oxidation, revealed that only two novel Bacteroides ovatus strains (D-6 and F-1), isolated from the faeces of healthy persons by TF-immunoaffinity enrichment, possessed structures that are immunochemically identical to the true TFα antigen. The TF-positive capsular polysaccharide structure of strain D-6 was characterized by mass spectrometry, monosaccharide composition analysis, glycosidase treatments and immunoblot staining with TFα- and TFβ-specific antibodies. The active antigen was identified as Galβ1-3GalNAc-, which was α-anomerically linked as a branching structure within a heptasaccharide repeating unit. We conclude that structures immunochemically identical to TFα are extremely rare on the surface of human intestinal bacteria and may only be identifiable by binding of both antibodies, NM-TF1 and NM-TF2, which recognize a complete immunomolecular imprint of the TFα structure. The two novel B. ovatus strains isolated in this study may provide a basis for the development of TF-based anti-tumor vaccines.     

Expression of the Clustered NeuAcα2–3Galβ O-Glycan Determines the Cell Differentiation State of the Cells

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2–3Galβ O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1–60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1–60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.