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The present paper reviews the occurrence of glycosidically bound aroma compounds in the plant kingdom and discusses different hypotheses concerning their role in plants. Emphasis is on biotechnological methods for flavor release and flavor enhancement through enzymatic hydrolysis of glycoconjugated aroma substances.
Human palatine tonsils are clinically important due to their susceptibility to tonsillitis and association with other local and systemic diseases. Paradoxically, the tonsils function as antigen sampling sites of the mucosal immune system and, consequently, the tonsil epithelia perform both protective and antigen sampling roles. These epithelia are divided into stratified squamous epithelium overlying the tonsil surface and crypt epithelium lining the tonsil crypts, the latter of which includes reticular areas which are infiltrated by lymphocytes and are responsible for antigen sampling. In this study we characterised cytokeratin and glycoconjugate expression by healthy epithelia of human palatine tonsils. We identified pan-epithelial tonsil markers and also demonstrated that the surface and reticular crypt epithelia are differentiated by the expression of multiple cytokeratins. The latter finding supports the hypothesis that these epithelia undergo alternate differentiation pathways and possess different functional roles. In addition, we identified cell subpopulations in the tonsil epithelia which may represent distinct cell subtypes including specialised antigen sampling cells. These findings establish a basis for future studies to investigate histochemical changes in tonsil epithelia that are associated with or predispose to local and/or systemic disease.
To clarify the origin of the central cells in hepatic erythroblastic islands, glycoconjugates on the surface of cellular constituents in fetal mice liver were ultrahistochemically examined using lectin staining. At 11 days of gestation, the cells derived from mesenchyme in fetal liver, including sinusoidal macrophages, endothelial cells, and erythropoietic cells, bound Griffonia simplicifolia isoagglutinin I-B4 (GS-I-B4), but hepatocytes lacked binding sites for the isolectin. Scavenger macrophages in the hepatic cords at 13 days of gestation and the central cells in the erythroblastic islands at 15 days of gestation also bound GS-I-B4. Hepatocytes, however, exhibited no GS-I-B4 binding site at any gestational day. At 11 days of gestation, none of the cells in fetal liver had binding sites for soybean agglutinin (SBA), but cells derived from mesenchyme acquired these binding sites at 13 days of gestation. The central cells in the erythroblastic islands also bound SBA, but hepatocytes did not bind the lectin at all. The central cells in the erythroblastic islands can be considered to belong to a mesenchymal cell lineage, and primitive sinusoidal macrophages at 11 days of gestation are possible precursors of these central cells.
Monoclonal antibodies and lectins were used to examine the expression patterns of apical membrane oligosaccharide sequences specific to type II pneumocytes in atypical adenomatous hyperplasia (AAH) and lung cancer. Atypical cells of AAH and papillary adenocarcinoma cells expressed abundant sialyl Thomsen-Friedenreich (TF) antigen: this was not observed in acinar adenocarcinoma, bronchioloalveolar carcinoma with mucin production or squamous cell carcinoma. Sialyl Tn antigens was also detected on a few cells in AAH and papillary adenocarcinomas. Asialo TF and Tn antigen were not observed on the surface of carcinoma cells of any type. Alpha(alpha)2,3-linked sialic acids predominated in type II pneumocyte, AAH and papillary adenocarcinoma, whereas ciliated columnar cells expressed alpha2,6-linked sialic acids. Lewisx and sialyl Lewisx antigens capped the TF antigen in both O- and N-linked side chains on the surface of AAH and papillary adenocarcinoma cells, but were not expressed by type II pneumocytes. The findings demonstrate that papillary adenocarcinoma cells resemble type II pneumocytes in that they express abundant sialyl TF surface antigen, but they also express TF-related antigens not found in type II pneumocytes. Apical surface glycoconjugates of AAH have structural characteristics shared by both type II pneumocytes and papillary adenocarcinoma cells.
The miracidia of Fasciola hepatica and Trichobilharzia ocellata approach their host snails Lymnaea truncatula and L. stagnalis by increasing their rate of change of direction (RCD) in increasing gradients of snail-conditioned water (SCW), and they perform a turnback swimming in decreasing gradients. Both hostfinding responses in both species were induced by glycoconjugates with a molecular weight of > 30 kDa that were sensitive to hydrolysis with pronase E and oxidation with NaIO4. Alkaline cleavage revealed that they contained carbohydrates linked O-glycosidically via serine and N-acetylgalactosamine. Miracidia clearly preferred SCW from their specific host snail versus other sympatric snail species and did not respond to water conditioned with fish, tadpoles, or leeches. Differences in the chemical characteristics of SCW from the intermediate hosts L. truncatula and L. stagnalis could be shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotting, and subsequent carbohydrate detection. The first step of purification of the effective signaling SCW components from both snail species was achieved by ion-exchange chromatography.
Cytochemical labeling with gold particle-conjugated lectins in combination with transmission and scanning electron microscopy was used to localise specific sugar residues on the Trichomonas vaginalis cell surface. For investigation of the role played by the surface glycoconjugates of T. vaginalis in the process of parasite adhesion to the target cells, selected glycan moieties of parasite surface-bound molecules were removed by treatment with alpha-mannosidase and beta-N-acetylglucosaminidase. For observation of the parasite/epithelial cell interaction, human amnion membrane was employed as an in vitro model. Ultrastructure observations showed that T. vaginalis has distinct binding sites for concanavalin-A and wheat-germ agglutinin. This indicates the presence of mannose or mannose-like residues and N-acetyl-D-glucosamine-containing residues on the parasite membrane. The addition of inhibitory sugars to T. vaginalis incubation media diminished the subsequent labeling of the parasite cell coat with lectins. Enzyme treatment caused a significant reduction in the number of sugar residues on the cell surface of the parasite. The majority of the viable, motile, enzyme-treated T. vaginalis cells incubated with amnion membrane were incapable of adhering to the target cells. It was concluded that sugar residues, in particular alpha-D-mannose and N-acetyl-glucosamine, present in the parasite glycocalyx are involved in the process of T. vaginalis attachment to the host's epithelial cells. Removal of the T. vaginalis cell-surface sugars prevented the attachment to and damage of the epithelial cells.
We have probed Tritrichomonas foetus, a protozoan parasite of the urogenital tract of cattle, with gold-labeled and fluorescent lectins, for the localization of glycoconjugates, at the cell surface and in internal cell compartments. The following lectins were used: Con A, PNA, WGA, BSI, BSII, HPA, WFA, SBA, LFA, HPA, and LCA. Carbohydrates were also localized using the Thiéry's technique. Carbohydrate residues were observed in the Golgi and vesicles with all lectins used. However, the labeling pattern varied among the cisternae. A high heterogeneity of cell labeling was detected in the same preparation. The nucleus and the nuclear envelope were labeled with Con A, WGA, BSI, and UEA I, suggesting the presence of D-Man, and/or D-glucose, L-fucose, and GlcNAc. Lysosomes were labeled with Con A and intensely labeled with HPA. The intensity of labeling of the plasma and flagellar membranes varied according to the lectin used.
Residualizing labels are radioactive or fluorescent tracers used for identifying the tissue and cellular sites in which circulating proteins are catabolized in the body. When attached to protein the labels do not affect normal mechanisms of protein catabolism, but remain at the cellular site of protein uptake, after the carrier protein itself is degraded to diffusible catabolites. Until recently these labels consisted of biologically indigestible carbohydrates attached to a radioactive reporter molecule. In this report we describe the synthesis and purification of a new fluorescent residualizing label, N,N-dilactitol-N'-fluoresceinyl-ethylenediamine. The label is prepared by first derivatizing ethylenediamine 1:1 with fluorescein isothiocyanate and then coupling lactose to the remaining primary amino group by reductive amination. A rapid one step purification of this and other glycoconjugate labels by reversed-phase high-pressure liquid chromatography is described.
A method for the determination of neutral sugars and hexosamines present in glycoconjugates by reverse-phase high-performance liquid chromatography (HPLC) of their phenylthiocarbamyl (PTC) derivatives has been developed. After acid hydrolysis, neutral sugars are converted to glycamines by reaction with ammonium acetate in the presence of sodium cyanoborohydride and are subsequently derivatized with phenylisothiocyanate, while the hexosamines present in the same hydrolysate, after separation on Dowex 50, are treated directly with this reagent. HPLC of the PTC-glycamines of the neutral sugars is performed on Microsorb C18 in an isocratic manner while chromatography of the PTC-hexosamines employs a Pico-Tag column with gradient elution to achieve separation from the PTC-amino acids. The procedure has proven to be highly sensitive, requiring as little as picomole amounts for the chromatographic step; monosaccharide compositions determined on glycoproteins and glycopeptides by this method were found to compare favorably to those previously obtained by other techniques.
This report examines the RP-HPLC separation of o-phthalaldehyde derivatives of amino acids, amino sugars, and amino sugar alcohols using either 2-mercaptoethanol or 3-mercaptopropionic acid. A method with pmol sensitivity for the analysis of N-acetylamino sugars of glycoconjugates was elaborated. Upon hydrolysis, amino sugars are reduced with borohydride. Automated precolumn derivatization and chromatographic conditions for the resulting hexosaminitols are the same as those used for the analysis of amino acids. The method has been tested with as little as 2 micrograms of bovine fetuin, with a glycopeptide from bromelain and with an oligosaccharide after periodate oxidation.
A microadhesion assay that allows the quantitative determination of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on 96-well polystyrene plates has been developed. After dislodging nonadherent cells by centrifugation, specifically bound cells are quantified by colorimetric analysis of a blue formazan product generated from the dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide by enzymatic reduction. Carbohydrate specificity of the cell adhesion was demonstrated by inhibition analyses and the general applicability of the assay was proved with indicator cells of three different origins: mouse fibrosarcoma cells, Chang liver cells, and human breast carcinoma cells (MDA-MB 231).
Traditional non-lectin staining methods and lectin histochemistry combined with specific exoglycosidase digestion were used to investigate the histological structure of these glands and to visualize the occurrence and composition of salivary glycoconjugates. The quail lingual glands comprise the anterior lingual gland, with rostral and caudal portions that differ in morphology and histochemical staining, and the posterior lingual gland devoid of regional differences. Carbohydrate histochemistry further differentiated the secretory structures by revealing a heterogeneous cell population of tubules within the rostral and caudal portions of the anterior gland, and a rather homogeneous staining of the tubuloalveolar elements in the rostral portion of the anterior lingual gland and in the posterior gland. Tubuloalveolar secretory cells mainly produced sulphate-containing molecules. In addition, sialoglycoconjugates were present in both anterior and posterior lingual glands, whereas fucoglycoconjugates were visualized only in the anterior gland.
Plasma membrane isolated by two-phase partition from rat liver showed rates of ascorbate free radical reduction by NADH of 4-5 nmoles of oxidized NADH/min/mg protein. This activity was inhibited 80% by ConA and up to 97% by WGA and LFA lectins. NADH-ascorbate free radical reductase was also inhibited in rat liver plasma membranes preincubated with neuraminidase or trypsin, but no additional inhibition was observed in the presence of LFA after enzyme digestion. It appears that the integrity of glucan moieities of the cell surface glycoconjugates are necessary for the optimal function of this activity that could be considered as part of the transplasma membrane electron transport system.
Lectin binding histochemistry was performed on the olfactory system of Pseudemys scripta to investigate the distribution and density of defined carbohydrate terminals on the cell surface glycoproteins of the olfactory receptors and their terminals in the olfactory bulbs. The lectin staining patterns indicate that the receptor cells of the olfactory mucosa are characterized by glycoconjugates containing alpha-D-galactose and N-acetyl-D-glucosamine terminal residues. The vomeronasal receptor cells contain instead alpha-N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and alpha-D-galactose residues. The results demonstrate that the vomeronasal receptor cells contain high density of alpha-N-acetyl-D-galactosamine sugar residues that are not expressed by receptor cells of the olfactory mucosa. The presence of specific glycoproteins, whose terminal sugars are detected by lectin binding, might be related to the chemoreception and transduction of the odorous message into a nervous signal or in the histogenesis of the olfactory system. In fact, the olfactory receptors are the only known neurons in the vertebrate nervous system that undergo a continual cycle of proliferation not only in developing animals but also in mature ones. Moreover the results show that BSA-I-B4, an alpha-D-galactosyl-specific isolectin, targets the terminal sugar residues in the ramified microglial cells.
The cellular ultrastructure and surface glycoconjugate expression of three life stages of Leishmania major were compared. Noninfective logarithmic phase promastigotes (LP) are immature cells bearing a thin cell coat, short flagellum, small and empty flagellar pocket, and a loose cytoplasm filled with profiles of ER and large Golgi complex. LP also contain subpopulations of maturing cells containing less ER and Golgi and synthesizing cytoplasmic granules of different size, number, and electron-density. Infective or metacyclic promastigotes (MP) are fully differentiated nondividing forms with a thickened, prominent cell coat, long flagellum, distended flagellar pocket filled with secretory material, and few cytoplasmic organelles other than abundant electron-dense granules. Tissue amastigotes also contain electron-dense cytoplasmic granules, their flagellar pockets are also enlarged and contain secretory material, but they lack a discernable cell coat. Immunogold labeling of GP63 on the cell surface was extensive only on amastigotes. Promastigote GP63 appeared to be masked by the presence of a densely packed lipophosphoglycan (LPG) coat which was extensively labeled on the entire surface of MP and LP. An elongated, developmentally modified form of LPG was abundantly labeled only on MP. LPG was poorly labeled on amastigotes, arguing that the promastigote cell coat is a stage-specific structure which is lost during intracellular transformation.
The glycosidic residues in the mandibular glands of five adult dogs were studied by using seven different lectin-horseradish peroxidase conjugates. In some cases a treatment with sialidase preceded the lectin staining. The mucous acinar cells contained oligosaccharides with alpha- and beta-N-acetylgalactosamine, N-acetylglucosamine and fucose residues, whereas the demilunar cells contained glycoconjugates rich in sialic acid linked to the penultimate disaccharide galactosyl-(beta 1-->3) N-acetylgalactosamine.
To determine whether nitric oxide (NO) regulates mucus secretion from airway submucosal glands which are the main source of human airway secretion, we examined the effects of NO synthase inhibitors (L-NAME and L-NMMA) on mucus glycoprotein (MGP) secretion from feline and human airway explants (with epithelium) and isolated submucosal glands. MGP secretion was estimated by measuring trichloroacetic-acid (TCA) precipitable [H3]-glycoconjugates using secretory indices. NO synthase inhibitors alone did not alter significantly MGP secretion from explants or isolated glands. Pretreatment with NO synthase inhibitors significantly inhibited both methacholine (MCh) and bradykinin (BK)-induced secretion from isolated glands, but not significantly inhibit the secretion from explants. The inhibition by L-NAME was reversed by the addition of L-arginine in both MCh- and BK-induced secretions from isolated glands. Further, a NO generator isosorbide dinitrate induced a significant increase in the secretion. These findings suggest that endogenous NO has a stimulatory action in airway submucosal gland secretion and directly regulates the secretion from submucosal glands independently of superficial epithelial cells.
Three different influenza virus neuraminidase (NA) genes have been subcloned into the vector pSC11 and expressed from the recombinant vaccinia viruses. These genes are from influenza viruses A/Tokyo/3/67 (N2); A/tern/Australia/G70c/75 (N9); and B/Hong Kong (HG)(NA of B/Lee/40). Cells infected with recombinants containing the NA gene express enzymatically active NA on the cell surface. The expressed protein results in the infected cells beings stripped of sialic acid, the receptor for influenza virus. This is not due to cleavage by NA from detached cells since at low multiplicity of infection only cells present at plaques are devoid of sialic acid. Thus NA is able to cleave sialic acid from neighboring glycoconjugates on the same membrane.