Cell Surface Glycoconjugates Research Articles

Carbohydrate receptor depletion as an antimicrobial strategy for prevention of urinary tract infection.

Many pathogenic microorganisms use host cell surface glycoconjugates, including glycosphingolipids (GSLs), as receptors [1, 2]. Pharmacologic agents that inhibit the biosynthesis of these receptors could therefore have a major impact on the clinical outcome of infection. N-alkylated imino sugars inhibit the first step in the GSL biosynthetic pathway [3, 4]. This step is catalyzed by a ceramide-specific glucosyltransferase, which transfers glucose to ceramide to form glucosylceramide (GlcCer), which is the precursor for all GlcCer-based neutral GSLs and gangliosides [5]. GSL biosynthesis inhibitors are potential therapeutics for the treatment of the GSL lysosomal storage diseases [6, 7]. Treatment with the glucose analogue N-butyldeoxynojirimycin (NBDNJ) caused 70% peripheral GSL depletion and was well tolerated in mice over a period of several months [8]. Efficacy was demonstrated in mouse GSL storage models, and clinical trials have been completed recently in type 1 Gaucher’s disease and are currently in progress in Fabry’s disease [9–11]. The drug NB-DNJ, which has undergone extensive animal and human testing, therefore offers a potential means of evaluating GSL depletion as an antimicrobial strategy. Uropathogenic Escherichia coli attach to epithelial cells through P fimbriae, which bind to Gala1-4Galb oligosaccharide sequences (globoseries) in cell surface GSLs [1]. We have evaluated the effects of GSL depletion on bacterial adhesion, cell activation, and in vivo infection.

Biosynthesis of N-acetylneuraminic acid in cells lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.

The first two steps in mammalian biosynthesis of N-acetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. A subclone of the human B lymphoma cell line BJA-B K20, lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains N-acetylmannosamine kinase activity. A gel filtration analysis of BJA-B K88 control cells, which express UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, revealed two N-acetylmannosamine kinase activity peaks, one co-eluting with UDP-N-acetylglucosamine 2-epimerase activity and one co-eluting with N-acetylglucosamine kinase. For this enzyme previous studies already showed a ManNAc kinase activity in vitro. In contrast, the hyposialylated BJA-B K20 subclone displayed only the N-acetylmannosamine kinase peak, co-migrating with N-acetylglucosamine kinase. The CMP-N-acetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supplementing the medium with N-acetylmannosamine. This N-acetylmannosamine-induced increase was drastically reduced by co-supplementation with N-acetylglucosamine only in K20 cells. We therefore propose the phosphorylation of N-acetylmannosamine as a hitherto unrecognized role of N-acetylglucosamine kinase in living cells.

Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs.

Many bacterial pathogens express lipooligosaccharides that mimic human cell surface glycoconjugates, enabling them to attach to host receptors and to evade the immune response. In Neisseria meningitidis, the galactosyltransferase LgtC catalyzes a key step in the biosynthesis of lipooligosaccharide structure by transferring alpha-d-galactose from UDP-galactose to a terminal lactose. The product retains the configuration of the donor sugar glycosidic bond; LgtC is thus a retaining glycosyltranferase. We report the 2 A crystal structures of the complex of LgtC with manganese and UDP 2-deoxy-2-fluoro-galactose (a donor sugar analog) in the presence and absence of the acceptor sugar analog 4'-deoxylactose. The structures, together with results from site-directed mutagenesis and kinetic analysis, give valuable insights into the unique catalytic mechanism and, as the first structure of a glycosyltransferase in complex with both the donor and acceptor sugars, provide a starting point for inhibitor design.

Ketone isosteres of 2-N-acetamidosugars as substrates for metabolic cell surface engineering.

Novel chemical reactivity can be engendered on cell surfaces by the metabolic incorporation of unnatural sugars into cell surface glycoconjuagtes. 2-N-Acetamido sugars such as GalNAc and GlcNAc are abundant components of cell surface glycoconjugates, and hence attractive targets for metabolic cell surface engineering. Here we report (1) the synthesis of isosteric analogs bearing a ketone group in place of the N-acetamido group, and (2) evaluation of their metabolic incorporation into mammalian cell surface glycans. A ketone isostere of GalNAc was metabolized by CHO cells through the salvage pathway and delivered to O-linked glycoproteins on the cell surface. Its residence at the core position of O-linked glycans is suggested by studies with a-benzyl GalNAc, an inhibitor of O-linked oligosaccharide extension. A mutant CHO cell line lacking endogenous UDP-GalNAc demonstrated enhanced metabolism of the GalNAc analog, suggesting that competition with native intermediates might limits enzymatic transformation in mammalian cells. A ketone isostere of GlcNAc could not be detected on CHO or human cell surfaces after incubation. Thus, the enzymes in the GlcNAc salvage pathway might be less permissive of unnatural substrates than those comprising the GalNAc salvage pathway. Alternatively, high levels of endogenous GlcNAc derivatives might compete with the ketone isostere andmore » prevent its incorporation into oligosaccharides.« less

O-linked N-acetylglucosamine and cancer: messages from the glycosylation of c-Myc.

Altered protein glycosylation is known to correlate with tumorigenesis, but its role remains enigmatic. Cells transformed by altered oncogene or tumor suppressor gene expression often also show changes of carbohydrate on cell surface glycoconjugates which correlate with the potential for tumor invasion and metastasis. In recent years, many oncogene and tumor suppressor gene products, such as c-Myc, SV40 large T antigen, and p53, were shown to be modified by O-GlcNAc. O-GlcNAc is a form of protein glycosylation found almost exclusively in the nucleus and cytoplasm of eukaryotic cells. The known O-GlcNAc-bearing proteins are phosphoproteins and form reversible multimeric complexes. O-GlcNAc modification is dynamic and appears to have a reciprocal relationship with protein phosphorylation. The enzymes which catalyze O-GlcNAc addition and removal have been characterized and used as effective tools in O-GlcNAc studies. It is of great interest in the future to investigate the alteration of O-GlcNAc in different cancers since addition/removal of O-GlcNAc on oncoproteins, tumor suppressor proteins, and other tumor-related proteins very likely plays a key role in the pathogenesis of tumors.

Carbohydrate expression in the intestinal mucosa.

The understanding of mechanisms responsible for alterations in mucin synthesis and secretions is essential in elucidating the aetiology of intestinal disorders. The existence of distinct mucins and M-cell transport mechanisms, and their beneficial effects, have long been recognised. Since nutritional and bacterial factors alter the mucin characteristics and are relevant to planning preventative strategies for intestinal diseases, the studies described in this monograph were designed to test the hypothesis that mucin composition and endocrine status of the intestinal tract are altered by dietary constituents and microbial flora. A study of gut-associated lymphoid tissue (GALT) was undertaken to test the hypothesis that lymphocyte-epithelial interactions influence the glycosylation of cells overlying Peyer's patches. The effects of diet and microbial flora were analysed by comparing the data from male Wistar germ-free rats, with conventional or human flora. Such rats were fed either a commercial diet, containing crude fibre, or a purified diet. Bone marrow transfers from syngeneic Balb/c mice to severe combined immunodeficient (SCID) mice were made to induce the formation of GALT. A comparison was made of the glycoconjugate profile of Peyer's patches in the guts of mice and humans. The results of the study showed that feeding a fibre-rich diet to germ-free rats resulted in increased villus-crypt lengths, decreased carboxylated mucin content of goblet cells, increased N-acetylglucosamine and sialic acid residues in the surface goblet cells, and a reduction in the number of endocrine cells in the small intestine. The lectin markers revealed M-cell development in the dome epithelium of the reconstituted SCID mice. Mouse M cells were labelled by fucose-specific lectins but in human Peyer's patches no distinct M-cell-staining pattern was observed. (1) the interactions between diet and flora alter the mucosal architecture and the activity of endocrine cells; (2) the dietary changes are influential in modifying the epithelial mucin predominantly in the small intestine while the microbial flora influences the mucosal architecture predominantly in the large intestine; (3) the use of bone marrow transplantation from syngeneic mice into SCID mice along with lectin markers for M cells can be used to study the histogenesis of Peyer's patches; (4) the distinct differences between mouse and human Peyer's patches suggest that when considering cell surface glycoconjugates as target molecules appropriate lectin should be used for each species. A future challenge in intestinal epithelial cell biology is identifying the nature and distribution of cell surface receptors for specific dietary components and bacteria.

A guide to the carbohydrate specificities of applied lectins-2 (updated in 2000).

The lectins, that can be used as tools to study glycobiological systems are defined as applied lectins (, , , , , , , , , , , , , ).They are easily obtained, stable and have their own binding specificity extending beyond the monosaccharide(, , , ,, , , , ).Their biochemical application has been reviewed extensively by Goldstein and Poretz (), Sharon and Lis () and Goldsteinet al. () Hundreds of lectins are used as applied lectins. Thus, organizing and grouping binding properties of these lectins should facilitate the selection of lectins as structural probes for studying glycans as well as the interpretation, distribution and properties of the carbohydrate chains on the cell surface. From the information provided by inhibition assays and binding properties, the carbohydrate specificities of applied lectins are classified into six groups according to their specificities to monosaccharides. The subgroups are based on lectin affinities to GalNAca1-0 to Ser(Thr) of the peptide chain, disaccharides, trisaccharides and, the number and the location of LFucal-’linked to oligosaccharides; all of these structures are found mainly in soluble glycoproteins and as cell surface glycoconjugates in mammals (, , , ,). Reviews concerning coding and classification of DGa1, GaINAc and Galf31-3/4G1cNAc specificities of applied lectins are given in Secion 1–4 of this proceeding () together with references , , , . A scheme of the classification is shown as follows.

Helicobacter pylori: A wolf in sheep's clothing: the glycotype families of Helicobacter pylori lipopolysaccharides expressing histo-blood groups: structure, biosynthesis, and role in pathogenesis

Publisher Summary This chapter focuses on the chemical structure, biosynthesis, and potential pathogenic role of the lipopolysaccharides (LPSs) from Helicobacter pylori . The structures of LPSs isolated from Helicobacter species found in nonhuman primates, which give rise to similar gastric symptoms in their respective hosts, are also described. LPSs are glycolipid structures carried by bacteria on their cell surfaces, which are actively involved in biochemical interactions between the bacterium and its host. The detection of LPSs in H. pylori expressing structures homologous to human histo-blood-group antigens stands as one of the most important and crucial discoveries in both H. pylori and bacterial-LPS research. The production of Lewis determinants, blood-group A, and linear blood-group B by H. pylori , occasionally by a single strain, is a perfect example of molecular mimicry, and of the capability that bacteria possess to elaborate glycan structures similar to those present in their biological niche. In this case, H. pylori carry LPSs with structures similar to cell surface glycolipids and glycoconjugates expressed by the human gastric mucosa. The chapter presents several reasons why H. pylori produce histo-blood-group antigens. One of the reasons is based on the molecular mimicry premise, in that H. pylori produces host-like gastric-glycan structures in order to mimic its immediate surrounding and thus avoid being detected by the host's immune system, leading to an uninterrupted long life.

A guide to monoclonal antibodies directed to glycotopes.

Monoclonal antibodies specific to glycotopes, the antigenic epitopes carried by glycoconjugates, have been widely utilized for study of the expression, distribution and function of carbohydrate determinants on a variety of cells and tissues (1,2).It is increasingly clear that cell surface glycoconjugates play important roles in cell-to-cell recognition and interaction (3,4).Functional analysis of glycotopes in such interaction has been greatly facilitated by the application of specific monoclonal antibodies. This chapter provides an introduction to the application of anti-glycotope antibodies in such analyses. For information regarding how to generate anti-carbohydrate antibodies, see refs. 1,2 or 5.

Altered glycosylation in inflammatory bowel disease: a possible role in cancer development.

Ulcerative colitis and Crohn's disease (together known as Inflammatory Bowel Disease or IBD) are both associated with increased risk for colorectal cancer. Although it is conventional to emphasise differences between IBD-associated and sporadic colon cancer, such as a lower rate of Adenomatosis Polyposis Coli mutations and earlier p53 mutations, IBD-associated cancer has a similar dysplasia-cancer sequence to sporadic colon cancer, similar frequencies of major chromosomal abnormalities and of microsatellite instability and similar glycosylation changes. This suggests that IBD-associated colon cancer and sporadic colon cancer might have similar pathogenic mechanisms. Because the normal colon is arguably in a continual state of low-grade inflammation in response to its microbial flora, it is reasonable to suggest that both IBD-associated and sporadic colon cancer may be the consequence of bacteria-induced inflammation. We have speculated that the glycosylation changes might result in recruitment to the mucosa of bacterial and dietary lectins that might otherwise pass harmlessly though the gut lumen. These could then lead to increased inflammation and/or proliferation and thence to ulceration or cancer. The glycosylation changes include increased expression of onco-fetal carbohydrates, such as the galactose-terminated Thomsen-Friedenreich antigen (Gal beta1,3GalNAc alpha-), increased sialylation of terminal structures and reduced sulphation. These changes cannot readily be explained by alterations in glycosyltransferase activity but similar changes can be induced in vitro by alkalinisation of the Golgi lumen. Consequences of these changes may be relevant not only for cell-surface glycoconjugates but also for intracellular glycoconjugates.

A novel subset of murine B cells that expresses unmasked forms of CD22 is enriched in the bone marrow: implications for B-cell homing to the bone marrow.

CD22 is a B-cell-restricted transmembrane protein, which acts as a negative regulator of B-cell signalling. CD22 also has lectin-like adhesive properties. When expressed on transfected fibroblasts, it is capable of mediating adhesion to other cells via recognition of cell-surface glycoconjugates terminating in alpha2,6-linked sialic acids. In previous studies in the mouse, CD22 was implicated as a bone marrow homing receptor for recirculating immunoglobulin D+ (IgD+) B cells through recognition of sialylated ligands on marrow sinusoidal endothelium. As the adhesive function of CD22 can be masked when alpha2,6-linked sialic acids are co-expressed at the cell surface, the aim of the present study was to investigate whether recirculating B cells have unmasked forms of CD22 that could be involved in bone marrow homing. Using alpha2,6-sialyllactose coupled to biotinylated polyacrylamide as a probe for detection of unmasked CD22, we showed that approximately 2-5% of IgD+ murine B cells in the spleen and mesenteric lymph nodes were able to bind this synthetic ligand. In the bone marrow, however, the fraction of IgD+ B cells with unmasked CD22 was increased by two- to fivefold. B cells from CD22-deficient mice were not stained with the polyacrylamide probe, confirming that staining of B cells in wild-type mice was caused by CD22 and not by other potential sialic acid-binding lectins. In conclusion, we have identified a new subset of mature B cells in the mouse with unmasked CD22. This subset of recirculating B cells may bind to CD22 ligands on bone marrow sinusoidal endothelium, leading to their selective homing and subsequent enrichment in this tissue.

Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells.

In this study we demonstrate that polysialyltransferases are capable of accepting unnatural substrates in terminally differentiated human neurons. Polysialyltransferases catalyze the glycosylation of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA). The unnatural sialic acid analog, N-levulinoyl sialic acid (SiaLev), was incorporated into cell surface glycoconjugates including PSA by the incubation of cultured neurons with the metabolic precursor N-levulinoylmannosamine (ManLev). The ketone group within the levulinoyl side chain of SiaLev was then used as a chemical handle for detection using a biotin probe. The incorporation of SiaLev residues into PSA was demonstrated by protection from sialidases that can cleave natural sialic acids but not those bearing unnatural N-acyl groups. The presence of SiaLev groups on the neuronal cell surface did not impede neurite outgrowth or significantly affect the distribution of PSA on neuronal compartments. Since PSA is important in neural plasticity and development, this mechanism for modulating PSA structure might be useful for functional studies.

The C-terminal N-glycosylation sites of the human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, adn -VI) are necessary for the expression of full enzyme activity.

The alpha1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

Cell surface glycoconjugates in the olfactory system of lungfish Protopterus annectens Owen

AbstractFranceschini, V. Lazzari, M. and Ciani, F. 2000. Cell surface glycoconjugates in the olfactory system of lungfish Protopterus annectens Owen. —Acta Zoologica (Stockholm) 81: 131–137Lectin binding was performed on the olfactory system of lungfish Protopterus annectens to identify specific glycoconjugates on the cell surface of olfactory receptor cells. The lectin histochemical patterns and the Western blot analysis indicate that the receptor cells of the olfactory mucosa are characterized by high density of α‐N‐acetyl‐ d‐galactosamine residues on the saccharidic chains of the surface glycoproteins. Other lectins display a regional pattern between the regions of the olfactory bulbs. This different histochemical lectin pattern might be due to a different regional segregation of the olfactory projections. On the other hand it could allow the identification of an area corresponding to the accessory olfactory bulb of terrestrial vertebrates in the ventrolateral region of Protopterus olfactory bulb. The presence in the dipnoan olfactory system of a vomeronasal organ homologous to the organ in amphibians is discussed. Moreover, the selective lectin binding on the surface of primary olfactory neurones suggests that specific cell surface glycoproteins may have a role in the axonal growth due to the continuous cycle of proliferation and the death of olfactory receptor cells.

Cell Surface Engineering by a Modified Staudinger Reaction

Selective chemical reactions enacted within a cellular environment can be powerful tools for elucidating biological processes or engineering novel interactions. A chemical transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chemically orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metabolism of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.

Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells.

Galectin-1 (gal-1) a member of the mammalian beta-galactoside-binding proteins recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45(+)Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca(2+)](i)) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca(2+)](i)is completely inhibited by lactose at 10 mM and was absent in CD45(-)Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca(2+)](i)stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca(2+)](i). Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase Cgamma1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs.

Oligosaccharides in Human Milk and Bacterial Colonization

Oligosaccharides in Human Milk and Bacterial Colonization

"Galectin-1 induces central and peripheral cell death: implications in T-cell physiopathology".

The immune system has a remarkable capacity to maintain a state of equilibrium even as it responds to a diverse array of foreign proteins and despite its contact exposure to self-antigens. Apoptosis is one of the mechanisms aimed at preserving the homeostasis after the completion of an immune response, thus returning the immune system to a basal state and warranting the elimination of autoagressive cells in both central and peripheral lymphoid organs. Targeted deletions in critical genes involved in the apoptotic death machinery together with natural spontaneous mutations have clearly shown the importance of apoptosis in the regulation of the immune response. This complex scenario of stimulatory and inhibitory genes has been enriched with the finding that galectin-1, a 14.5 kDa β-galactoside-binding protein, is able to induce apoptosis of immature cortical thymocytes and mature T cells by cross-linking cell surface glycoconjugates. Galectin-1 is present not only in central and peripheral lymphoid organs, but also at sites of immune privilege. In the present article we will discuss the implications of galectin-1-induced apoptosis in T-cell physiopathology in an attempt to validate its therapeutic potential in autoimmune and inflammatory diseases.

Overview

This symposium entitled, "Bacterial Colonization of the Gut and the Use of Pre- and Probiotics" was organized by the Shanghai Children's Medical Center of the Second Shanghai Medical University in conjunction with the Combined Program in Pediatric Gastroenterology and Nutrition at Harvard Medical School to underscore a 10-year agreement between both medical centers to provide training of academic pediatricians from China in basic and translational research. The training program is supported by a training grant from the National Institute of Child Health and Development (NICHD)/Fogarty Center (1-U01A145441-01), and research projects are, in part, supported by a research grant from Wyeth Nutritionals International, Inc., (Philadelphia, PA, U.S.A.) The symposium, itself, was sponsored from an educational grant from Wyeth Nutritionals International, Inc. for which the the organizers are grateful. The topic for this initial Sino-American symposium represents an important area of research in pediatrics and a major research interest of the two organizing groups. Dr. W. Allan Walker, a pediatric gastroenterologist with a strong research interest in the development of host defense, introduces the topic of the symposium by considering the importance of pre- and probiotics in the development of intestinal host defense in the extrauterine environment. He reviews the effects of initial colonization of the gut on the development of the effector immune response to antigen stimulation. At birth, the gut's lymphoid tissue has the capacity to respond to microbial-food antigens. However, the initial colonization of the newborn gut with indigenous microflora provides the appropriate "adjuvant effect" to activate antibody production and T-cell activation. Furthermore, the nature of initial oral feedings (breast-milk vs. formula) helps to determine the nature of colonizing bacteria (lactobacilli and bifidobacteria vs. enterobacteria and Escherichia coli). In addition, specific nutrients such as nucleotides, omega-3 fatty acids, and lactoferrin can enhance the effects of indigenous flora on strengthening the mucosal barrier and minimizing inflammatory responses to pathologic bacteria colonization. Dr. David S. Newburg, a glycobiologist from Harvard Medical School, reviews the importance of oligosaccharides in human milk on the attachment of pathologic bacteria to glycoconjugates and glycolipids and proteins in the intestinal microvillus membrane. Oligosaccharides, a natural prebiotic, has an influence on the nature of colonizing bacteria. He notes that enterocyte cell surface glycoconjugates are pathogen receptors and help to influence pathologic colonization of the newborn gut. In contrast, soluble oligosaccharides in breast milk can attach to "adhesions" on pathogens and thereby block their interaction with the intestinal surface and reduce their colonization. He cites examples of breast milk protection by oligosaccharides for Campylobacter and rotaviral infection. These studies may help to identify additives to future infant formula. Dr. Michael S. Caplan, a neonatologist from North-western University with a strong research interest in necrotizing enterocolitis, and Dr. Tamas Jilling reviews the possible role of probiotics in the treatment of necrotizing enterocolitis. The immature human enteric surface has an immature glycosylation pattern favoring colonization with pathologic organisms. In the premature neonate, this process may contribute to the pathogenesis of necrotizing enterocolitis. If probiotics are introduced into the diet of the newborn at birth, their presence could block pathologic colonization and thereby reduce the incidence of necrotizing enterocolitis. The authors provide evidence in an experimental model for necrotizing enterocolitis that the use of probiotics (Lactobacillus GG) can prevent the upregulation of platelet-activating factor, an inflammatory cytokine and indicator of early necrotizing enterocolitis. Dr. Dingwei Dai, an investigator at the Shanghai Children's Medical Center and a research fellow in the Developmental Gastroenterology Laboratory of the Combined Program in Pediatric Gastroenterology and Nutrition at Harvard Medical School, and colleagues from the United States present exciting new data to support the notion that glycosylation of microvillus membrane lipids and protein through glycosyltransferase activity is developmentally regulated and that cortisone treatment of pregnant or newborn animals can activate the maturation of this process. They also provide preliminary evidence that glycosylation in the human fetus is also under developmental regulation. These observations help explain pathologic colonization of the human newborn and the potential for treatment of this process with growth factors such as cortisone. Finally, Dr. Jon A. Vanderhoof, a pediatric gastroenterologist from the University of Nebraska Medical Center, reviews the use of probiotics in the treatment inflammatory disorders in infants and children. He reviews clinical studies to suggest that Lactobacillus GG administered orally to children who have rotaviral infection reduces the severity and length of time of the clinical illness. He also reviews its use in the treatment of allergic dermatitis of children and its preliminary use in the treatment of inflammatory bowel disease. These presentations provide an excellent review of the current state of our knowledge of the role of nutrients in bacterial colonization in health and disease of infants and children.

Palate development. In vitro procedures.

The mammalian secondary palate provides an excellent experimental paradigm with which to investigate regulation of the sequential expression and interaction of molecular signals during embryogenesis. The secondary palate arises as bilateral extensions of the oral aspect of the maxillary processes of the first arch. In mammalian embryos, these palatal extensions make contact, fuse with one another and give rise to the secondary palate (roof of the oral cavity) (,). The palatal processes, consisting of mesenchymal cells embedded in a loosely organized extracellular matrix and enclosed within a stratified epithelium, initially grow vertically on either side of the tongue. The processes subsequently undergo a series of morphogenetic movements that result in their reorientation above the tongue, thereby bringing the medial edge epithelium (MEE), on the apical surface of each process, into contact forming a midline epithelial seam. Cells of the MEE undergo a precise sequence of molecular changes that culminate in removal of the midline epithelial seam. These changes include cessation of DNA synthesis, increased synthesis of cell-surface glycoconjugates, increased synthesis of lysosomal enzymes, cell death of the peridermal (surface) cell layer, and transdifferentiation of the subjacent MEE from an epithelial to a mesenchymal phenotype. Fusion of the palatal processes, followed by peridermal cell death and transdifferentiation of the MEE and migration into the underlying mesenchyme results in formation of the definitive secondary palate (,).