Effects of dietary forage-to-concentrate ratio and forage type on milk phospholipids and fatty acid composition of polar lipids.

The effects of grass silage and red clover silage on milk fatty acid (FA) composition are extensively studied, but little is known of their effects on minor lipid constituents of milk fat globule membrane. We investigated the effects of forage:concentrate (FC) ratio in grass silage-based diets and forage type (grass silage vs. red clover silage) on selected molecular species of milk phospholipids (PL) and the FA composition of PL. Ten multiparous Nordic Red cows were offered following dietary treatments: grass silage-based diets containing 70:30 (HG) or 30:70 (LG) FC ratio or a red clover silage-based diet (RC) comprising 50:50 FC ratio on a dry matter basis. The most abundant molecular species within the phosphatidylcholines was 16:0-18:1 phosphatidylcholine that was increased by 18% in HG compared with LG milk. Dietary treatments did not affect the relative proportion of 18:1-18:1+18:0-18:2 phosphatidylethanolamine that was the most prevalent species (ca. 44%-45%) in that class. We identified the d18:1-22:0 sphingomyelin as the most abundant sphingomyelin species that tended to increase in HG milk compared with LG. The FC ratio did not affect the relative proportions of saturated FA nor monounsaturated FA in PL, but the proportion of cis-9 18:1 was elevated in HG versus LG milk, whereas the proportion of 18:2n-6 was 50% higher in LG versus HG milk. The RC diet increased monounsaturated FA and 18:3n-3 levels in PL compared with grass silage-based diets and decreased the relative proportion of saturated FA. However, the RC diet did not affect the relative proportion of polyunsaturated FA in PL, although red clover silage typically increases the proportion of polyunsaturated FA in milk fat. This study provides valuable knowledge of the minor lipid components in milk on species level in relation to common feeding strategies in high-forage systems.

The effect of dietary forage to concentrate ratio and forage type on milk fatty acid composition and milk fat globule size of lactating cows.

We examined the effects of 2 grass silage-based diets differing in forage:concentrate (FC) ratio and those of a red clover silage-based diet on intake, milk production, ruminal fatty acid (FA) biohydrogenation, milk FA composition, and milk fat globule (MFG) size distribution. Ten multiparous Nordic Red cows received the following treatments: grass silage-based diets containing high (70:30, HG) or low (30:70, LG) FC ratio or a red clover silage-based diet with an FC ratio of 50:50 (RC) on a dry matter basis. Determinations of MFG were performed from fresh milk samples without addition of EDTA so the results of fat globules >1 µm in diameter are emphasized instead of the entire globule population. Lower FC ratio in grass silage-based diets increased milk production with no effect on daily fat yield, leading to 13% lower milk fat concentration. The effect of FC ratio on MFG size was moderate. It did not affect the volume-weighted diameter in grass silage-based diets, although LG lowered the volume-surface diameter of MFG in the size class >1 µm compared with HG. Compared with HG, feeding LG moderately decreased the biohydrogenation of 18:2n-6, leading to a higher level of polyunsaturated fatty acids in milk fat. Feeding RC lowered milk fat concentration and daily milk fat yield compared with grass silage-based diets. The volume-weighted diameter of MFG in the size class >1 µm was smaller in RC milk compared with grass silage-based diets. Feeding RC increased the flow of 18:3n-3 at the omasum by 2.4-fold and decreased the apparent ruminal 18:3n-3 biohydrogenation compared with grass silage-based diets despite similar intake of 18:3n-3. It also resulted in the lowest amount of saturated FA and the highest amounts of cis-9 18:1, 18:3n-3, and polyunsaturated FA in milk. In conclusion, LG decreased milk fat content and induced minor changes in MFG size distribution compared with HG, whereas RC lowered milk fat production, altered milk FA composition to nutritionally more beneficial direction, and led to smaller MFG compared with grass silage-based diets.

Breast milk IgA to foods has different epitope specificity than serum IgA-Evidence for entero-mammary link for food-specific IgA?

BACKGROUND: We have previously shown that maternal cow's milk (CM) elimination results in downregulation of CM-specific IgA antibody levels in BM, but not in serum, suggesting that an entero-mammary link may exist for food-specific antibody-secreting cells. OBJECTIVE: We sought to investigate whether food-specific IgA epitope profiles differ intra-individually between mother's serum and BM. We also examined how infants' food epitope-specific IgA develops in early infancy and the relationship of IgA epitope recognition with development of cow's milk allergy (CMA). METHODS: We measured specific IgA to a series of overlapping peptides in major CM allergens (αs1 -, αs2 -, ß- and κ-caseins and ß-lactoglobulin) in paired maternal and infant serum as well as BM samples in 31 mother-infant dyads within the first 15 post-partum months utilizing peptide microarray. RESULTS: There was significant discordance in epitope specificity between BM and maternal sera ranging from only 13% of sample pairs sharing at least one epitope in αs1 -casein to 73% in κ-casein. Epitope-specific IgA was detectable in infants' sera starting at less than 3 months of age. Sera of mothers with a CMA infant had increased binding of epitope-specific IgA to CM proteins compared to those with a non-CMA infant. CONCLUSION & CLINICAL RELEVANCE: These findings support the concept that mother's milk has a distinct antifood antibody repertoire when compared to the antibody repertoire of the peripheral blood. Increased binding of serum epitope-specific IgA to CM in mothers of infants with CMA may reflect inherited systemic immunogenicity of CM proteins in these families, although specific IgA in breast milk was not proportionally up-regulated.

Demodex gatoi-associated contagious pruritic dermatosis in cats--a report from six households in Finland.

BACKGROUND: Demodex gatoi is unique among demodectic mites. It possesses a distinct stubby appearance, and, instead of residing in the hair follicles, it dwells in the keratin layer of the epidermis, causing a pruritic and contagious skin disease in cats. Little is known of the occurrence of D. gatoi in Europe or control of D. gatoi infestation. CASE PRESENTATION: We describe D. gatoi in 10 cats, including five Cornish Rex, two Burmese, one Exotic, one Persian and one Siamese, living in six multi-cat households in different locations in Finland containing 21 cats in total. Intense pruritus was the main clinical sign. Scaling, broken hairs, alopecia and self-inflicted excoriations were also observed. Diagnosis was based on finding typical short-bodied demodectic mites in skin scrapings, skin biopsies or on tape strips. Other pruritic skin diseases, such as allergies and dermatophytoses, were ruled out. In one household, despite finding several mites on one cat, all six cats of the household remained symptomless. Amitraz used weekly at a concentration of 125-250 ppm for 2-3 months, proved successful in three households, 2% lime sulphur weekly dips applied for six weeks in one household and peroral ivermectin (1 mg every other day for 10 weeks) in one household. Previous trials in four households with imidacloprid-moxidectin, selamectin or injected ivermectin given once or twice a month appeared ineffective. CONCLUSION: D. gatoi-associated dermatitis is an emerging contagious skin disease in cats in Finland. Although pruritus is common, some cats may harbour the mites without clinical signs. In addition, due to translucency of the mites and fastidious feline grooming habits, the diagnosis may be challenging. An effective and convenient way to treat D. gatoi infestations has yet to emerge.

Detection of circulating tumour cells in peripheral blood with an automated scanning fluorescence microscope.

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.

Pelodera (syn. Rhabditis) strongyloides as a cause of dermatitis--a report of 11 dogs from Finland.

BACKGROUND: Pelodera (Rhabditis) strongyloides is a small saprophytic nematode that lives in decaying organic matter. On rare occasions, it can invade the mammalian skin, causing a pruritic, erythematous, alopecic and crusting dermatitis on skin sites that come into contact with the ground. Diagnosis of the disease is based on case history (a dog living outdoors on damp straw bedding) with characteristic skin lesions and on the demonstration of typical larvae in skin scrapings or biopsy. Pelodera (rhabditic) dermatitis cases have been reported mainly from Central European countries and the United States. CASE PRESENTATION: During 1975-1999, we verified 11 canine cases of Pelodera dermatitis in Finland. The cases were confirmed by identifying Pelodera larvae in scrapings. Biopsies for histopathology were obtained from three cases, and typical histopathological lesions (epidermal hyperplasia, epidermal and follicular hyperkeratosis, folliculitis and furunculosis with large numbers of nematode larvae of 25-40 microm of diameter within hair follicles) were present. The Pelodera strongyloides dermatitica strain from the first verified case in Finland has been maintained in ordinary blood agar in our laboratory since 1975. Light microscopy (LM) and scanning electron microscopy (SEM) studies were employed to obtain detailed morphological information about the causative agent. The rhabditiform oesophagus at all developmental stages, the morphology of the anterior end of the nematode, copulatory bursa and spicules of the male and the tail of the female were the most important morphological features for identifying P. strongyloides. CONCLUSION: These cases show that Pelodera dermatitis occurs in Finland, and also farther north than described earlier in the literature. This condition should be considered when a dog living outdoors has typical skin lesions situated at sites in contact with the ground as the main presenting clinical feature. The fastest and easiest way to confirm the diagnosis is to demonstrate typical larvae in skin scrapings. In uncertain cases, skin biopsy and culturing of the worms are recommended as supplementary diagnostic procedures.

Gluten-free diet may alleviate depressive and behavioural symptoms in adolescents with coeliac disease: a prospective follow-up case-series study.

BACKGROUND: Coeliac disease in adolescents has been associated with an increased prevalence of depressive and disruptive behavioural disorders, particularly in the phase before diet treatment. We studied the possible effects of a gluten-free diet on psychiatric symptoms, on hormonal status (prolactin, thyroidal function) and on large neutral amino acid serum concentrations in adolescents with coeliac disease commencing a gluten-free diet. METHODS: Nine adolescents with celiac disease, aged 12 to 16 years, were assessed using the semi-structured K-SADS-Present and Lifetime Diagnostic interview and several symptom scales. Seven of them were followed at 1 to 2, 3, and 6 months on a gluten-free diet. RESULTS: Adolescent coeliac disease patients with depression had significantly lower pre-diet tryptophan/ competing amino-acid (CAA) ratios and free tryptophan concentrations, and significantly higher biopsy morning prolactin levels compared to those without depression. A significant decrease in psychiatric symptoms was found at 3 months on a gluten-free diet compared to patients' baseline condition, coinciding with significantly decreased coeliac disease activity and prolactin levels and with a significant increase in serum concentrations of CAAs. CONCLUSION: Although our results of the amino acid analysis and prolactin levels in adolescents are only preliminary, they give support to previous findings on patients with coeliac disease, suggesting that serotonergic dysfunction due to impaired availability of tryptophan may play a role in vulnerability to depressive and behavioural disorders also among adolescents with untreated coeliac disease.

Contagious mucocutaneous dermatitis of the mountain hare (Lepus timidus): pathology and cause.

Contagious mucocutaneous dermatitis is a frequently encountered disease of mountain hares (Lepidus timidus) in Finland. We describe the histopathologic changes and propose an etiologic cause for this disorder. Fifty-three cases collected during 1982-2000 were examined histologically. Transmission electron microscopy was performed in one case. In fully developed lesions, keratinocytes in epidermis and follicular infundibula were swollen and contained large eosinophilic intracytoplasmic inclusion bodies with marked reticular and ballooning degeneration. In later stages, there was marked necrosis and ulceration with severe pyogranulomatous and suppurative inflammation. At this stage, no viral inclusions were detectable, but secondary Staphylococcus warnerii infection was present in most cases. In late lesions, there was dermal fibrosis with epidermal hyperplasia. No spiral-shaped bacteria suggesting treponematosis were detected at any stage. Ultrastructurally, swollen epidermal and follicle infundibular cells contained round intracytoplasmic inclusion bodies with a myriad of virions typical of poxvirus with a biconcave nucleocapsid core, two lateral bodies, and a clearly discernible outer lipoprotein capsule. The findings suggest that contagious mucocutaneous dermatitis in mountain hares is a viral disease caused by a poxvirus. The disease is often complicated by secondary bacterial infection, most commonly S. warneri.

Function and structure of Drosophila glycans.

Through the application of classic organismal genetic strategies, such as mutagenesis and interaction screens, Drosophila melanogaster provides opportunities to understand glycan function. For instance, screens for Drosophila genes that establish dorsal-ventral polarity in the embryo or that influence cellular differentiation through signal modulation have identified putative glycan modifying enzymes. Other genetic and molecular approaches have demonstrated the existence of phylogenetically conserved and novel oligosaccharide processing activities and carbohydrate binding proteins. While the structural characterization of Drosophila oligosaccharide diversity has lagged behind the elucidation of glycan function, landmarks are becoming apparent in the carbohydrate terrain. For instance, O-linked GlcNAc and mucins, spatially and temporally regulated N-linked oligosaccharide expression, glycosphingolipids, heparan sulfate, chondroitin sulfate and polysialic acid have all been described. A major challenge for Drosophila glycobiology is to expand the oligosaccharide structural database while endeavoring to link glycan characterization to functional analysis. The completion of the Drosophila genome sequencing project will yield a broad portfolio of glycosyltransferases, glycan modifying enzymes and lectins requiring characterization. To this end, the great range of genetic tools that allow the controlled spatial and temporal expression of transgenes in Drosophila will permit unprecedented manipulation of glycosylation in a whole organism.

Zwitterionic and acidic glycosphingolipids of the Drosophila melanogaster embryo.

Defining glycosphingolipid structures in species amenable to genetic manipulation, such as Drosophila melanogaster, provides a foundation for investigating mechanisms that regulate glycolipid expression. Therefore, eight of the 12 major glycosphingolipids, accounting for 64% of lipid-linked carbohydrate in Drosophila embryos, were purified after separation into acidic and zwitterionic pools. The zwitterionic lipids possess phosphoethanolamine (PEtn) linked to one or more GlcNAc residues and comprise a family of serially related structures. The longest characterized glycolipid, an octaosylceramide, designated Nz28, has the structure: GalNAcbeta, 4(PEtn-6)GlcNAcbeta,3Galbeta,3GalNAcalpha,4Ga lNAcbeta, 4(PEtn-6)GlcNAcbeta,3Manbeta,4GlcbetaCer. Heptaosyl (Nz7), hexaosyl (Nz6), pentaosyl (Nz5) and tetraosyl (Nz4) forms of Nz28, sequentially truncated from the nonreducing terminus, possess only one PEtn moiety. The major acidic lipid, designated Az29, possesses two PEtn moieties and a glucuronic acid linked to a Gal-extended Nz28. Two other acidic glycolipids, Az9 and Az6, exhibit one PEtn moiety and the same hexose and N-acetylhexosamine composition as Az29 and Nz6, respectively. The fully extended Drosophila core oligosaccharide differs from that of other dipterans in the linkage at a single glycosidic bond, a distinction with significant structural and biosynthetic consequences. Furthermore, acidic species account for a larger proportion of total glycosphingolipid, and PEtn substitution of GlcNAc is more complete in the Drosophila embryo. Divergent characteristics may reflect interspecies variation or stage-specific glycosphingolipid expression in dipterans.

Enzymatic transfer of a beta1,6-linked N-acetylglucosamine to the alpha-galactose of globo-N-tetraose: in vitro synthesis of a novel hybrid pentasaccharide of lacto-globo type.

A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and UDP[3H]GlcNAc with hog gastric mucosal microsomes, known to contain beta1,6-N-acetylglucosaminyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAc beta1-3([3H]GlcNAc beta1-6)Gal alpha1-4Gal beta1-4Glc. The new enzyme activity possesses substrate specificity features common to a purified beta1,6-GlcNAc-transferase from bovine tracheal epithelium, which forms branches at the subterminal beta1,3-substituted galactose and accepts both GlcNAc- and Gal-configuration at the terminal residue of the acceptor (Ropp et al. (1991) J. Biol. Chem., 266, 23863-23871). The new beta1,6-GlcNAc-branch was readily galactosylated by bovine milk beta1,4-galactosyltransferase, revealing a pathway to novel hybrid type glycans with N-acetyllactosamine chains on globotype saccharides. This pathway may lead to the rare IP blood-group antigen and to globoside-like molecules mediating cell adhesion.

Synthesis of a tetravalent sialyl Lewis x glycan, a high-affinity inhibitor of L-selectin-mediated lymphocyte binding to endothelium.

Kidney transplant rejection is an inflammatory process characterized by lymphocyte infiltration. Our earlier observations have shown that peritubular capillary endothelium (PTCE) is the site of lymphocyte entry into the rejecting renal allograft. During rejection, PTCE begins to express sialyl Lewis x de novo, and binds lymphocytes by a mechanism largely dependent on L-selectin. Hence, inhibiting the lymphocyte-endothelial interaction with oligosaccharide ligands of L-selectin offers an attractive possibility to prevent the inflammation and rejection. Here, we report enzyme-assisted synthesis of N-acetyllactosamine-based tetra-, deca-, and docosameric saccharides carrying one, two or four distally located sialyl Lewis x groups [Neu-NAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc] (sLex), respectively. When tested for their ability to inhibit lymphocyte-endothelial interaction during rat kidney transplant rejection, all sLex-saccharides were inhibitors in the Stamper-Woodruff binding assays; the analogues lacking fucose showed no inhibitory potency. The tetravalent sLex glycan proved to be a high-affinity adhesion inhibitor with an IC50 < 50 nM. While less powerful than the tetravalent glycan, also the divalent sLex saccharide was a much better inhibitor than the monovalent glycan. Hence, increasing multivalency and, possibly, increasing chain length of the polylactosamine backbone, enhances the inhibitory potency of sLex bearing glycans in the lymphocyte-endothelial adhesion assay. This suggests that L-selectin behaves as a "functional oligomer" on lymphocyte surfaces.

De novo expression of endothelial sialyl Lewis(a) and sialyl Lewis(x) during cardiac transplant rejection: superior capacity of a tetravalent sialyl Lewis(x) oligosaccharide in inhibiting L-selectin-dependent lymphocyte adhesion.

Acute organ transplant rejection is characterized by a heavy lymphocyte infiltration. We have previously shown that alterations in the graft endothelium lead to increased lymphocyte traffic into the graft. Here, we demonstrate that lymphocytes adhere to the endothelium of rejecting cardiac transplants, but not to the endothelium of syngeneic grafts or normal hearts analyzed with the in vitro Stamper-Woodruff binding assay. Concomitant with the enhanced lymphocyte adhesion, the cardiac endothelium begins to de novo express sialyl Lewis(a) and sialyl Lewis(x) (sLea and sLex) epitopes, which have been shown to be sequences of L-selectin counterreceptors. The endothelium of allografts, but not that of syngeneic grafts or normal controls, also reacted with the L-selectin-immunoglobulin G fusion protein, giving further proof of inducible L-selectin counterreceptors. The lymphocyte adhesion to endothelium could be significantly decreased either by treating the lymphocytes with anti-L-selectin antibody HRL-1, or by treating the tissue sections with sialidase or anti-sLea or anti-sLex monoclonal antibodies. Finally, we synthetized enzymatically several members of the sLex family oligosaccharides and analyzed their ability to block lymphocyte adhesion to cardiac endothelium. The monovalent sLex (a tetramer), divalent sLex (a decamer), and tetravalent sLex (a 22-mer) could all significantly reduce lymphocyte binding, but the inhibition by the tetravalent sLex-construct was clearly superior to other members of the sLex family. The crucial control oligosaccharides, sialyl lactosamines lacking fucose but being otherwise similar to the members of sLex family, had no effect on lymphocyte binding.

Enzyme-assisted synthesis of a bivalent high-affinity dodecasaccharide inhibitor of mouse gamete adhesion. The length of the chains carrying distal alpha 1,3-bonded galactose residues is critical.

Proposing to study the molecular mechanisms of mouse gamete adhesion with the aid of high affinity adhesion inhibitors of saccharide nature, we report here the enzymatic synthesis of a bivalent oligosaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), consisting of two long arms that link together two distal alpha 1,3-galactose residues. Binding data reported elsewhere (E. Litscher et al., Biochemistry, 1995, 34, 4662-4669) show that 4 is a high affinity inhibitor of mouse gamete adhesion in vitro (IC50 = 9 microM), while a related octasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, consisting of two short arms is of very low inhibitory activity. The data highlight the importance of the two alpha-galactose residues of 4, and the length of the sugar chains joining them.

Oligosaccharide constructs with defined structures that inhibit binding of mouse sperm to unfertilized eggs in vitro.

During fertilization in mice, free-swimming sperm bind to mZP3, an 83-kDa glycoprotein present in the egg extracellular coat, the zona pellucida [Wassarman, P. M. (1990) Development 108, 1-17]. Mouse sperm recognize and bind to a specific class of serine/threonine-linked (O-linked) oligosaccharides present on mZP3. After binding to mZP3, sperm undergo a form of cellular exocytosis, the acrosome reaction, thereby enabling them to penetrate the zona pellucida and fertilize the egg. Thus, gamete interactions in mice are carbohydrate-mediated. In this context, we tested 15 O-linked-related oligosaccharide constructs with defined structures for their ability to inhibit binding of mouse sperm to ovulated eggs and to induce sperm to undergo the acrosome reaction in vitro. Thirteen of the oligosaccharides were constructed and characterized in our laboratory [Seppo, A., Pentillä, L., Niemelä, R., Maaheimo, H., Renkonen, O., & Keane, A. (1995) Biochemistry 34, 4655-4661]; two were obtained commercially. We found that, while none of the oligosaccharides induced sperm to undergo the acrosome reaction, a few of them inhibited binding of sperm to eggs at relatively low concentrations (ID50 < 5 microM). In certain cases, sperm formed head-to-head aggregates in the presence of the oligosaccharides. The results suggest that the ability of oligosaccharides to inhibit binding of sperm to eggs is dependent on several parameters, including the size and branching pattern of the oligosaccharide, as well as on the nature of the sugar residue at the nonreducing end of the oligosaccharide.

Enzymatic synthesis of octadecameric saccharides of multiply branched blood group I-type, carrying four distal alpha 1,3-galactose or beta 1,3-GlcNAc residues.

Radiolabeled oligosaccharide constructs were prepared to evaluate carbohydrate determinants involved in gamete adhesion in mice. The octasaccharide primer GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (1) was incubated with UDP-GlcNAc and beta 1,6-GlcNAc-transferase of hog gastric microsomes, producing the tetraantennary decasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (2). The decasaccharide was then incubated with UDP-Gal and beta 1,4-galactosyltransferase from bovine milk, yielding the tetradecasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (3). Incubation of the tetradecasaccharide 3 with UDP-Gal and alpha 1,3-galactosyltransferase from bovine thymus gave the octadecameric glycan Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (4).(ABSTRACT TRUNCATED AT 250 WORDS)

Stepwise transfer of alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc sequences to 3-OH and 6-OH of distal galactose residues in bi-, tri-, and tetra-antennary asialo-glycans of N-linked complex type.

The hydroxyl groups 3 and 6 of distal galactose units in bi-, tri-, and tetra-antennary asialo-glycans of N-linked complex type were substituted stepwise by transferase reactions with the sequence alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc. The products of each transferase reaction were purified chromatographically and the structures were confirmed by 1H NMR spectroscopy. Molecular weights of the final products were determined by matrix-assisted laser-desorption mass spectrometry (MALDI-MS).

Enzymatic synthesis of two lacto-N-neohexaose-related Lewis x heptasaccharides and their separation by chromatography on immobilized wheat germ agglutinin.

Radiolabelled lacto-N-neohexaose was fucosylated with partially purified alpha (1,3)fucosyltransferase(s) from human milk. Structural analysis of the monofucosylated products obtained at an early stage of the reaction revealed that both distal branches of the acceptor had reacted equally well, generating Lewis x determinants, while the reducing end glucose had not reacted. The two isomeric Lewis x glycans were readily separated from each other by chromatography on immobilized wheat germ agglutinin (WGA), because alpha (1,3)fucosylation of the (1 --> 6)-linked branch of lacto-N-neohexaose was associated with a dramatic loss of WGA affinity. The fucosylation mixture of lacto-N-neohexaose also contained a difucosylderivative that carried Lewis x determinants at both distal branches. Attempted refucosylation of this octasaccharide failed to transfer fucose to the glucose unit.

Bi-antennary oligo-(N-acetyllactosamino)glycans of I-type are galactosylated preferentially at the GlcNAc beta 1-6Gal linked arms by alpha 1,3-galactosyltransferase of bovine thymus.

alpha 1,3-Galactosylation of radiolabelled bi-antennary acceptors Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal-R (R = 1-OH, beta 1-4GlcNAc or beta 1-4Glc) with bovine thymus alpha 1,3-galactosyltransferase was studied. At all stages of the reactions the three acceptors reacted faster at the 1-->6 linked arm than at the 1-->3 linked branch. Hence, in addition to the doubly alpha 1,3-galactosylated products, practically pure Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal-R could be obtained from the three acceptors in reactions that had proceeded to near completion. The isomeric mono-alpha 1,3-galactosylated products were identified by using exoglycosidases to remove the branches unprotected by alpha 1,3-galactoses and by subsequently identifying the resulting linear glycans chromatographically.

A novel beta-N-acetylglucosaminidase activity in hog gastric mucosal microsomes: preferential hydrolysis of terminal GlcNAc beta 1-3 linkages in GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, but GlcNAc beta 1-6 linkages in GlcNAc beta 1-3(GlcNAc beta 1-6)Gal.

Hog gastric mucosal microsomes contain beta-N-acetylglucosaminidase activity which cleaves GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc at the terminal GlcNAc beta 1-3Gal linkage faster than at the GlcNAc beta 1-6Gal bond, producing mainly GlcNAc beta 1-6Gal beta 1-4GlcNAc. In a marked contrast, GlcNAc beta 1-3(GlcNAc beta 1-6)Gal is cleaved primarily at the GlcNAc beta 1-6Gal bond, while partial hydrolysis of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc reveals similar rates of cleavage for the (1-3) and (1-6) linkages. Our data support the notion that the terminal beta 1,6-linked GlcNAc unit of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc may interact with the reducing end GlcNAc unit intramolecularly in water solution.