Impact of Daily Grazing Time on Dairy Cow Welfare-Results of the Welfare Quality® Protocol.

Grazing provides livestock better opportunities to act out their species-specific behavior compared to restrictive stable conditions. The aim of the present study was to examine the effects of daily grazing time on welfare of dairy cows in organic and conventional farms based on the Welfare Quality® assessment protocol for dairy cattle (WQ®). Therefore, we applied the WQ® on 32 dairy farms (classified in 3 groups: Group 0, minor/zero grazing, n = 14; Group 1, medium grazing, n = 10; Group 2, high grazing, n = 8). We assessed the status of animal welfare once in winter and once in summer. For statistical analyses we used mixed models for repeated measures, with group, season, and their interaction as fixed factors. At the WQ® criteria level, five out of nine examined criteria improved in farms with grazing between winter and summer. In contrast, the welfare situation in minor/zero grazing farms remained largely unchanged. At the level of WQ® measures, only the individual parameters "% of cows with hairless patches" and "% of lame cows" were affected positively by high grazing. Grazing offers a potential to enhance welfare of dairy cows during the summer season, while beneficial effects are not guaranteed when management does not satisfy the animals´ needs.

Site directed processing: role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2.

BACKGROUND: The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 beta3-Gal-transferase (C1GalT) and core 2 beta6-GlcNAc-transferase (C2GnT). Beta4-Gal-transferase (beta4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates. METHODS: We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6. RESULTS: Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the +3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galbeta1-3GalNAcalpha-TAGV was identified as an efficient C2GnT substrate. CONCLUSIONS: The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion. GENERAL SIGNIFICANCE: Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.

Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat.

The cell membrane mucin MUC1 is over-expressed and aberrantly glycosylated in many cancers, and cancer-associated MUC1 glycoforms represent potential targets for immunodiagnostic and therapeutic measures. We have recently shown that MUC1 with GalNAcalpha1-O-Ser/Thr (Tn) and NeuAcalpha2-6GalNAcalpha1-O-Ser/Thr (STn) O-glycosylation is a cancer-specific glycoform, and that Tn/STn-MUC1 glycopeptide-based vaccines can override tolerance in human MUC1 transgenic mice and induce humoral immunity with high specificity for MUC1 cancer-specific glycoforms (Sorensen AL, Reis CA, Tarp MA, Mandel U, Ramachandran K, Sankaranarayanan V, Schwientek T, Graham R, Taylor-Papadimitriou J, Hollingsworth MA, et al. 2006. Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology. 16:96-107). In order to further characterize the immune response to Tn/STn-MUC1 glycoforms, we generated monoclonal antibodies with specificity similar to the polyclonal antibody response found in transgenic mice. In the present study, we define the immunodominant epitope on Tn/STn-MUC1 glycopeptides to the region including the amino acids GSTA of the MUC1 20-amino acid tandem repeat (HGVTSAPDTRPAPGSTAPPA). Most other MUC1 antibodies are directed to the PDTR region, although patients with antibodies to the GSTA region have been identified. A panel of other MUC1 glycoform-specific monoclonal antibodies was included for comparison. The study demonstrates that the GSTA region of the MUC1 tandem repeat contains a highly immunodominant epitope when presented with immature short O-glycans. The cancer-specific expression of this glycopeptide epitope makes it a prime candidate for immunodiagnostic and therapeutic measures.

UDP-Gal: GlcNAc-R beta1,4-galactosyltransferase--a target enzyme for drug design. Acceptor specificity and inhibition of the enzyme.

Galactosyltransferases are important enzymes for the extension of the glycan chains of glycoproteins and glycolipids, and play critical roles in cell surface functions and in the immune system. In this work, the acceptor specificity and several inhibitors of bovine beta1,4-Gal-transferase T1 (beta4GalT, EC 2.4.1.90) were studied. Series of analogs of N-acetylglucosamine (GlcNAc) and GlcNAc-carrying glycopeptides were synthesized as acceptor substrates. Modifications were made at the 3-, 4- and 6-positions of the sugar ring of the acceptor, in the nature of the glycosidic linkage, in the aglycone moiety and in the 2-acetamido group. The acceptor specificity studies showed that the 4-hydroxyl group of the sugar ring was essential for beta4GalT activity, but that the 3-hydroxyl could be replaced by an electronegative group. Compounds having the anomeric beta-configuration were more active than those having the alpha-configuration, and O-, S- and C-glycosyl compounds were all active as substrates. The aglycone was a major determinant for the rate of Gal-transfer. Derivatives containing a 2-naphthyl aglycone were inactive as substrates although quinolinyl groups supported activity. Several compounds having a bicyclic structure as the aglycone were found to bind to the enzyme and inhibited the transfer of Gal to control substrates. The best small hydrophobic GlcNAc-analog inhibitor was found to be 1-thio-N-butyrylGlcNbeta-(2-naphthyl) with a K(i) of 0.01 mM. These studies help to delineate beta4GalT-substrate interactions and will aid in the development of biologically applicable inhibitors of the enzyme.

Tooth loss treatment in the anterior region: autotransplantation of premolars and cryopreservation.

Avulsed and lost anterior teeth are common in young people. Using autotransplantation, it is possible to move problems in dental arches to regions where they are more easy to solve orthodontically. Transplantation of premolars with three-quarter root formation or full root formation with wide-open apical foramina provides the best prognosis for long-term survival. This article describes the use of autotransplantation and orthodontic treatment, together with cryopreservation, in connection with complicated trauma in the anterior region of an 8-year-old girl.

Electron capture dissociation of O-glycosylated peptides: radical site-induced fragmentation of glycosidic bonds.

Glycosylation of proteins represents one of the most important post-translational modifications. The structural characterisation of glycoproteins--especially with respect to the determination of the glycosylation site--by direct mass spectrometric methods still remains an elusive goal. We have applied the low energy dissociation method electron capture dissociation (ECD) in a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer to the structural elucidation of mucin-derived peptides glycosylated with glycans of different core types. Capture of an electron by multiply protonated precursor ions [M + nH](n+) resulted in the formation of reduced odd electron radical cations [M + nH](n-1)+*. Subsequent cleavage of the N-Calpha bonds of the peptide chain, mostly without loss of the labile sugar moiety, represents a major fragmentation pathway allowing unambiguous assignment of the glycosylation site. In addition to peptide backbone cleavages, loss of acetyl radicals from the N-acetyl group of the HexNAc glycans is observed. Radical site induced elimination processes of the glycan moieties initiated by hydrogen transfer, from the glycan to the peptide backbone and vice versa give rise to signals in the ECD spectra. The different sugar core types exhibit different fragmentation patterns driven by the stability of the resulting fragments allowing the discrimination of isomeric glycans.

Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1).

Glycosylation determines essential biological functions of epithelial mucins in health and disease. We report on the influence of glycosylation of the immunodominant DTR motif of MUC1 on its antigenicity. Sets of novel glycopeptides were synthesized that enabled us to examine sole and combined effects of peptide length (number of repeats) and O-glycosylation with GalNAc at the DTR motif on the binding patterns of 22 monoclonal antibodies recognizing this motif. In case of unglycosylated peptides almost all antibodies bound better to multiple MUC1 tandem repeats. Glycosylation at the DTR led to enhanced binding in 11 cases, whereas 10 antibodies were not influenced in binding, and one was inhibited. In nine of the former cases both length and DTR glycosylation were additive in their influence on antibody binding, suggesting that both effects are different. Improved binding to the glycosylated DTR motif was exclusively found with antibodies generated against tumor-derived MUC1. Based on these data a tumor-specific MUC1 epitope is defined comprising the ...PDTRP... sequence in a particular conformation essentially determined by O-glycosylation at its threonine with either GalNAcalpha1 or a related short glycan. The results can find application in the field of MUC1-based immunotherapy.

A glycopeptide in complex with MHC class I uses the GalNAc residue as an anchor.

Peptides bind MHC class I molecules by anchoring hydrophobic side chains into pockets in the peptide binding groove. Here, we report an immunogenic (in vitro and in vivo) MUC1 glycopeptide (MUC1-8-5GalNAc) bound to H-2Kb, fully crossreactive with the nonglycosylated variant. Molecular modeling showed that the central P5-Thr-GalNAc residue points into the C pocket and forms van der Waals and hydrogen bond interactions with the MHC class I. As predicted, GalNAc, a modified peptide carrying an additional anchor in the central C anchor pocket, increased the affinity by approximately 100-fold compared with the native low-affinity peptide (MUC1-8). The findings demonstrate that glycopeptides associated with MHC class I molecules can use GalNAc to anchor the peptide in the groove and enable high-affinity binding.

Use of synthetic oligosaccharide substrate analogs to map the active sites of N-acetylglucosaminyltransferases I and II.

Tables III and IV summarize substrate analog data presented in Tables I and II, respectively. Data for GlcNAc-T I shown in Tables I and III correlate very well with the crystal structure for GlcNAc-T I. This indicates that substitution of the various hydroxyl groups by hydrogen, [table: see text] O-methyl, and maybe even larger O-alkyl groups does not cause appreciable changes to either the overall conformation of the oligosaccharide or the binding mode, thus supporting this approach of chemical modification of oligosaccharide substrates for mapping of the binding site. There is as yet no crystal structure for GlcNAc-T II. These studies indicate both advantages and disadvantages of this approach for elucidating the catalytic and binding sites of an enzyme. Substrate analog data indicate which chemical groups in the substrate are essential for catalysis and binding and suggest the type of linkage involved (hydrogen bond donor or acceptor). However, no information has been obtained on the protein groups involved in these interactions. If a crystal structure is available, the substrate analog conclusions are primarily confirmatory. However, whether or not a crystal structure is available, this approach can be very helpful in the design of specific inhibitors.

Conformational studies on the MUC1 tandem repeat glycopeptides: implication for the enzymatic O-glycosylation of the mucin protein core.

The tandem repeat of the MUC1 protein core is a major site of O-glycosylation that is catalyzed by several polypeptide GalNAc-transferases. To define structural features of the peptide substrates that contribute to acceptor substrate efficiency, solution structures of the 21-residue peptide AHGVTSAPDTRPAPGSTAPPA (AHG21) from the MUC1 protein core and four isoforms, glycosylated with alpha-N-acetylgalactosamine on corresponding Thr residues, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5,T17), were investigated by NMR spectroscopy and computational methods. NMR studies revealed that sugar attachment affected the conformational equilibrium of the peptide backbone near the glycosylated Thr residues. The clustering of the low-energy conformations for nonglycosylated and glycosylated counterparts within the VTSA, DTR, and GSTA fragments (including all sites of potential glycosylation catalyzed by GalNAc-T1, -T2, and -T4 transferases) showed that the glycosylated peptides display distinct structural propensities that may explain, in part, the differences in substrate specificities exhibited by these polypeptide GalNAc-transferases.

Complex carbohydrates are not removed during processing of glycoproteins by dendritic cells: processing of tumor antigen MUC1 glycopeptides for presentation to major histocompatibility complex class II-restricted T cells.

In contrast to protein antigens, processing of glycoproteins by dendritic cells (DCs) for presentation to T cells has not been well studied. We developed mouse T cell hybridomas to study processing and presentation of the tumor antigen MUC1 as a model glycoprotein. MUC1 is expressed on the surface as well as secreted by human adenocarcinomas. Circulating soluble MUC1 is available for uptake, processing, and presentation by DCs in vivo and better understanding of how that process functions in the case of glycosylated antigens may shed light on antitumor immune responses that could be initiated against this glycoprotein. We show that DCs endocytose MUC1 glycopeptides, transport them to acidic compartments, process them into smaller peptides, and present them on major histocompatability complex (MHC) class II molecules without removing the carbohydrates. Glycopeptides that are presented on DCs are recognized by T cells. This suggests that a much broader repertoire of T cells could be elicited against MUC1 and other glycoproteins than expected based only on their peptide sequences.

NMR-based determination of the binding epitope and conformational analysis of MUC-1 glycopeptides and peptides bound to the breast cancer-selective monoclonal antibody SM3.

Mucin glycoproteins on breast cancer cells carry shortened carbohydrate chains. These partially deglycosylated mucin 1 (MUC-1) structures are recognized by the monoclonal antibody SM3, which is being tested for its diagnostic utility. We used NMR spectroscopy to analyze the binding mode and the binding epitope of peptide and glycopeptide antigens to the SM3 antibody. The pentapeptide PDTRP and the glycopentapeptide PDT(O-alpha-D-GalNAc)RP are known ligands of the monoclonal antibody. The 3D structures of the ligands in the bound conformation were determined by analyzing trNOESY build-up rates. The peptide was found to adopt an extended conformation that fits into the binding pocket of the antibody. The binding epitopes of the ligands were determined by saturation transfer difference (STD) NMR spectroscopy. The peptide's epitope is predominantly located in the N-terminal PDT segment whereas the C-terminal RP segment has fewer interactions with the protein. In contrast, the glycopeptide is interacting with SM3 utilizing all its amino acids. Pro1 shows the strongest binding effect that slightly decays towards Pro5. The GalNAc residue interacts mainly via the N-acetyl residue while the other protons show less interactions similar to that of Pro5. The glycopeptide in the bound state also has an extended conformation of the peptide with the carbohydrate oriented towards the N-terminus. Docking studies showed that peptide and glycopeptide fit the binding pocket of the mAb SM3 very well.

Three-dimensional magnetic resonance image of the mandible and masticatory muscles in a case of juvenile chronic arthritis treated with the Herbst appliance.

The present report documents, in a case of juvenile chronic arthritis (JCA) with mandibular retrognathia, three-dimensional (3D) changes in the mandible and the relationship between the mandible and the masticatory muscles resulting from treatment with the Herbst appliance after cessation of growth. Magnetic resonance scanning of the whole head was carried out before and after treatment. The mandible, the masseter, and the medial and lateral pterygoid muscles were segmented bilaterally and reconstructed in 3D for both stages. Superimposition of the datasets was carried out according to anatomical structures in the brain (cranial base). Mandibular superimposition was performed according to the mandibular symphysis and the lower mandibular border. The mandible moved forward and downward relative to the anterior cranial base. In addition, bone apposition was observed at the superior and posterior surfaces of both mandibular condyles and at the roof of the glenoid fossa. The masticatory muscles remained relatively stable in position in relation to the anterior cranial base. To our knowledge, such information in JCA patients has not previously been published in the literature. Using magnetic resonance imaging (MRI), it was possible to gain improved insight into the 3D morphology including soft tissues without the overlap of the surrounding tissues observed in the conventional radiographs. Accordingly, it is suggested that 3D magnetic resonance analysis is a more useful method for the follow-up of the JCA patients than radiographic techniques.

Glycoprotein biosynthesis in porcine aortic endothelial cells and changes in the apoptotic cell population.

Porcine aortic endothelial cells (PAECs) produce glycoproteins with important biological functions, such as the control of cell adhesion, blood clotting, blood pressure, the immune system, and apoptosis. Cell surface glycoproteins play important roles in these biological activities. To understand the control of cell surface glycosylation, we elucidated biosynthetic pathways leading to N- and O-glycans in PAECs. Based on the enzyme activities, PAECs should be rich in complex biantennary N-glycans. In addition, the enzymes synthesizing complex O-glycans with core 1 and 2 structures are present in PAECs. The first enzyme of the O-glycosylation pathway, polypeptide GalNAc-transferase, was particularly active. Its specificity toward synthetic peptide substrates was found to be similar to that of purified bovine colostrum enzyme T1. A significant fraction of PAECs treated with tumour necrosis factor alpha or human serum detached from the culture plate, and most of these cells were apoptotic. The apoptotic cell population exhibited decreased core 2 beta 6-GlcNAc-transferase activity. In contrast, the activities of core 1 beta 3-Gal-transferase, which synthesizes O-glycan core 1, and of alpha 3-sialyltransferase (O), which sialylates core 1, were increased in apoptotic PAECs. Thus, apoptotic PAECs are predicted to have fewer complex O-glycans and a higher proportion of short, sialylated core 1 chains.