Ebner's Glands Research Articles

Physico-chemical characterization of human von Ebner gland protein expressed in Escherichia coli: implications for its physiological role.

The human von Ebner gland protein (VEG) was expressed in Escherichia coli and purified to homogeneity. The sequence and mass of the recombinant protein were confirmed, and far and near UV circular dichroic analyses showed that the protein was properly folded. The secondary structure of recombinant VEG consisted of 75% beta-sheets and 12% alpha-helices, and it was found to be stable under acidic conditions, in the presence of alcohol, and at high temperatures. The denaturation temperature was 79 degreesC at pH 3.5, with a denaturation enthalpy (DeltaHd) of 160,600 J/mol. Fluorescence analysis and measurement of the denaturation temperature by circular dichroism did not detect any interaction between VEG and extremely bitter (denatonium benzoate, caffein) or sweet (aspartame) compounds. These results suggest that VEG may not function as a shuttle for transfer of sapid molecules to taste receptors.

Expression of a Lipocalin in Human Nasal Mucosa

A 19 kDa soluble protein was purified from human nasal mucus. Its N-terminal amino-acid sequence appeared to be identical to that of a lipocalin synthesised both in lachrymal glands and in von Ebner's glands (VEG) of circumvallate papillae. In order to verify whether this protein was synthesised in the nasal cavity or was the result of tear contamination, we adopted an immunohistochemical approach. Polyclonal antibodies, raised against a primate VEG protein, were used on sections of human nasal mucosa obtained from surgery. The results clearly indicate that the protein is synthesised in sero-mucous glands underlying the respiratory ciliated epithelium. Although ligand-binding experiments with some odorant molecules have given negative results, we cannot exclude a role of odorant solubiliser and carrier for this protein.

The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms.

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.

Ultrastructure of Weber's salivary glands of the root of the tongue in the rat.

The mammalian tongue encompasses several sizeable agglomerations of minor salivary glands. The ultrastructure of the various glands in the body of that organ has already been determined. In contrast, almost nothing is known of the structure of Weber's glands, a collection of salivary glands in the root of the tongue. The entire tongue was extirpated from anesthetized adult male rats that had been perfused with fixative and specimens of tissue that included Weber's glands were dissected from the lingual root. These were prepared for electron microscopic examination by conventional means. Weber's glands in the rat are mixed glands, consisting of long mucous tubules that often are capped by serous demilunes. There appear to be no ducts per se, the mucous tubules increasing in caliber and approaching the crypts of the dorsal lingual surface while still retaining their mucous secretory character. As these ducts near the surface, their lining changes to stratified squamous epithelium. The mucous cells are cytologically similar to those in the sublingual glands of the same animal. Mucous droplets undergo vertical fusion to form centrally situated intracellular channels through which mucus is exocytosed, much in the same manner as goblet cells. The serous cells, which have all of the hallmarks of protein-secreting cells, probably empty their secretory granules via cellular extensions that directly reach the tubule lumen since there are no intercellular canaliculi between adjacent mucous cells. The mucus elaborated by Weber's glands undoubtedly aids in swallowing dry food. We postulate that the serous cells in these glands, as in the more anterior von Ebner's glands, might play a role in the mechanism of taste, especially where posteriorly situated, nonlingual taste buds are concerned.

Structure and organization of the porcine LCN1 gene encoding Tear lipocalin/von Ebner's gland protein

We have cloned and sequenced the entire genomic fragment of the porcine LCN1 gene which encodes Tear lipocalin/von Ebner's gland protein, a member of the lipocalin superfamily highly expressed in porcine lachrymal and lingual glands. The porcine LCN1 gene is approximately 4.6 kb in size and contains six protein-coding exons and a 3′-nontranslated exon. The structure of this porcine gene is highly similar, in terms of numbers of exons/introns, in size of exons and in intron phasing, to that of the human LCN1 and rat VEGP genes, thus supporting a very close evolutionary relationship of these genes. Within the promoter region of the porcine LCN1 a putative TATA box, a CAAT box and two MRE motifs are found. The same MRE motifs are conserved in the human LCN1 promoter, suggesting that they might be of relevance for LCN1 gene expression. However, additional motifs present in the human LCN1 promoter, such as AP-1 and AP-2 sites, a NF- κB site, and a cAMP-responsive element, could not be detected in the porcine LCN1 promoter.

Ultrastructure of the salivary glands in the midtongue of the common vampire bat, Desmodus rotundus.

All examined mammals have at least two sets of lingual salivary glands: von Ebner's glands and Weber's glands. A third set, the glands of Blandin and Nuhn, is present in the tongues of some but not all mammals. Vampire bats, Desmodus rotundus, are unusual in that they possess another set of lingual glands, these being in the midtongue region. The anterior half of the tongue was extirpated from several adult vampire bats, dissected, and tissue blocks derived from the midregions of the body of the tongue prepared for transmission electron microscopy by conventional means. The midlingual glands are in the form of long, tubular secretory endpieces that are succeeded by ducts of simple morphology. In general, the secretory portions consist of two cell types, which may be intermingled in the same tubule or may form tubules that consist wholly of one cell type or the other. Seromucous cells usually have one or several rough endoplasmic reticulum cisternae that are hugely distended by a homogeneously dense material. Their granules have a bizonal substructure: one or several dense bands are embedded in a lighter matrix. Mucous cells are rather typical in structure, but their secretory product is different from run-of-the-mill mucous droplets. These droplets vary in density from cell to cell. In some cells, these droplets have a relatively light matrix; in other cells, the droplet is unusually dense, consisting mainly of a dark, structureless matrix with marginal lenticular lacunae of low density in which some short, irregular filaments are scattered. A rare finding is the presence of ciliated cells intermingled with secretory endpiece cells. The cilia are of conventional morphology. Secretory tubules are succeeded by ducts that resemble intercalated ducts; the epithelium of these ducts gradually increases in height to form a kind of excretory duct, without the intervention of striated ducts. As the ducts approach the lingual surface, the epithelium changes to stratified squamous. Saliva produced by the midlingual glands may be an aid in the reciprocal grooming behavior of vampire bats. Based on their morphology, the excurrent ducts may not modify the initial saliva elaborated by these glands and might act simply as pipelines by which the saliva reaches the mouth.

Basic and lectin histochemical characterization of bovine gustatory (von Ebner's) glands.

The Bovine tongue possesses numerous circumvallate papillae (8-16 each side). The troughs around the papillae are the openings of the ducts of the gustatory (von Ebner's) glands. In this study, we have characterized in situ the glycosidic composition of the secretion of bovine gustatory glands using traditional histochemical methods and lectin histochemistry with and without prior neuraminidase (sialidase) digestion. The lectin-horseradish peroxidase conjugates employed were: PNA, DBA, SBA, WGA, LTA, UEA I and ConA. Acinar cells show a diffuse positivity towards PAS and Alcian blue at pH 2.5 and the most intense and homogeneous lectin staining was obtained with PNA. This indicates that bovine gustatory glands secrete glycoproteins with 1,2-glycol containing hexoses and carboxyl-rich glycoconjugates and that galactosyl (beta 1-->3) Nacetylgalactosamine is the most frequent sugar residue present in these glycoproteins. Results were compared with data reported in the literature on the same glands of other species.

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: identification of the catalytically active serine, aspartic acid, and histidine residues

Human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) constitute a family of mammalian lipases characterized by an acidic pH optimum. HGL and RLL are secreted by the chief cells of the stomach and by the serous von Ebner's glands of the tongue, respectively, and hydrolyze dietary longchain triglycerides in the gastrointestinal tract. HLAL, in contrast, catalyzes the intralysosomal degradation of both triglycerides and cholesteryl esters in virtually all cells except erythrocytes. All three enzymes are proposed to be serine esterases with a catalytic Ser-Asp-His triad similar to other lipases, despite their sensitivity towards sulfhydryl modifying reagents. To investigate the role of conserved serine, aspartic acid, and histidine residues in HLAL and HGL, we constructed 24 mutant lipases with single amino acid substitutions using the site-directed mutagenesis approach. Our combined data strongly support the conclusion that Ser153, Asp324, and His355 are components of the catalytic triad of HLAL and HGL. Structural integrity of the conserved His-Gly dipeptide of lipases also appears to be important for neutral lipid hydrolysis, as replacement of His65 by glutamine abolished HLAL and HGL enzymic activity. Substitution of HLAL residues Asp93, Asp130, and Asp328 with glycine, in contrast, had a more pronounced impact on cholesteryl oleate hydrolysis than on triglyceride hydrolysis. These results provide new insights into the structural basis of HLAL and HGL function.

The acid lipase gene family: three enzymes, one highly conserved gene structure

Human gastric lipase (HGL; triacylglycerol lipase; EC 3.1.1.3) plays an important role in the digestion of dietary triglycerides in the gastrointestinal tract, especially in patients suffering from pancreatic lipase deficiencies. The enzyme is secreted by the fundic mucosa of the stomach and hydrolyzes the ester bonds of triglycerides under acidic pH conditions, while cholesteryl esters are not attacked. The 379-amino acid protein is highly homologous to two other acidic lipases, rat lingual lipase (RLL; triacylglycerol lipase; EC 3.1.1.3) and human lysosomal acid lipase (HLAL; cholesteryl esterase; EC 3.1.1.13). To determine whether this remarkable similarity is also present at the genomic level, we have elucidated the respective gene structures by screening three bacteriophage lambda libraries and by polymerase chain reaction-based intron amplification. The genes encoding HGL, RLL, and HLAL are composed of 10 exons interrupted by nine introns and span about 14 kb, 18.7 kb, and 38.8 kb of genomic DNA, respectively. The HGL and RLL gene organizations are identical, suggesting that RLL is the rat gastric lipase expressed in the serous von Ebner glands of the tongue. The positions of the HLAL intervening sequences are also absolutely conserved, except for the location of intron 1. Our results support the concept that HLAL and HGL/RLL are members of a gene family of lipases that most likely have evolved by duplication of an ancestral gene and subsequently assumed distinct roles in neutral lipid metabolism due to sequence divergence and different expression patterns.

Effect of Substance P and Receptor Antagonists on Secretion of Lingual Lipase and Amylase from Rat von Ebner's Gland

Field, R. B., S. J. Chirtel and R. S. Redman. Effect of substance P and receptor antagonists on secretion of lingual lipase and amylase from rat von Ebner's gland. Peptides 18(2) 277–285, 1997.— Substance P (SP, 1 μM) when incubated with minced von Ebner's glands for 15, 30, and 60 min, stimulated secretion of lingual lipase (12.14%±0.90) and amylase (8.30%±0.42). Only 10 μM of the SP receptor antagonist CP-96,345 significantly inhibited SP-evoked secretion. d-Pro2-d-Phe7-d-Trp9-SP (Ia), d-Pro2-d-Trp7,9-SP (Ib), d-Arg1-d-Trp7,9-d-Leu11-SP (Ic), or 1 μM CP-96,345 were not effective, suggesting that the SP receptor of von Ebner's gland might be an isoform. Propranolol and timolol, β1/β2-adrenergic receptor antagonists were not effective and the cholinergic receptor antagonist, atropine, was effective in only slightly reducing amylase secretion but not lingual lipase. Differential secretion of the two enzymes was observed for basal and stimulated secretion. Thus, exocytosis may not be the only pathway involved in SP-evoked protein secretion.

Peptidergic Innervation in Human von Ebner's Glands: An Immunohistochemical Study

Matsuda H, Kusakabe T, Kohno H, Nagahara T, Furukawa M, Sawada H, Kawakami T, Takenaka T, Tsukuda M Peptidergic innervation in human von Ebner's glands: an immunohistochemical study. Acta Otolaryngol (Stockh) 1997; 117: 447-451.The occurrence and distribution of several neuropeptides were studied in human von Ebner's glands. Immunoreactivity for substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), galanin, and somatostatin was found in the nerve fibers around the acini, ducts, and blood vessels. VIP-immunoreactive varicose fibers were numerous compared with the other five neuropeptides. Most NPY fibers were associated with the vasculature in the gland. These findings suggest that the neuropeptides may regulate the secretion and vascular tone in human von Ebner's glands.

Freeze-substitution and Postembedding Immunocytochemistry on Rat Taste Buds: G-Proteins, Calcitonin Gene-related Peptide, and Choline Acetyl Transferase

Abstract: We have explored freeze-substitution combined with low-temperature embedding in rat taste buds for postembedding immunocytochemistry. A major difference in taste bud cells that were rapidly frozen without prior chemical fixation and those that were fixed and cryoprotected before freezing was that electron-dense core granules were virtually absent. The antibodies used in these initial studies were directed against calcitonin gene-related peptide (CGRP), a peptide commonly found in nociceptive neurons; the α-subunits of two G-proteins involved in bitter and sweet taste transduction; and choline acetyl transferase (ChAT), an enzyme involved in the synthesis of acetylcholine. Anti-CGRP immunolabeled a subpopulation of unmyelinated perigemmal neurons; anti-Gqα labeled a larger subpopulation of these neurons and the microvilli of cells that were most likely from Type II vallate taste buds. α-Gustducin was found in cytoplasm of Type II and/or III cells and probably in microvilli of Type I cells of vallate taste buds. The best labeling results were obtained with anti-ChAT, which stained microvilli and lateral membranes of some Type II vallate taste bud cells, and the cytoplasm of some other Type II and/or III vallate cells. In addition, anti-ChAT labeled electron-opaque materials inside taste bud pores of vallate papillae, but, under the same conditions, not granules of Type I cells or most of the vesicles in von Ebner's glands. These data suggest that we can not assume a priori that the contents of the electron-dense core granules of Type I cells, or even of those of von Ebner's glands, contain the precursors of the taste bud pore–dense substances.

The Salivary Lipocalin Von Ebner's Gland Protein Is a Cysteine Proteinase Inhibitor

The lipocalins make up a heterogeneous superfamily of proteins. Although showing almost no sequence homology, they share very similar secondary and tertiary structures. Their ability to bind hydrophobic ligands is well established, but the physiological function of most lipocalins remains unclear. The lipocalin from the human Von Ebner's Gland of the tongue (VEGh) contains three sequence motifs corresponding with the papain-binding domains of cystatins, a family of naturally occurring cysteine proteinase inhibitors. We found that VEGh inhibited papain activity to a similar extent as salivary cystatin S. Furthermore, synthetic peptides derived from VEGh and cystatin C, comprising these three motifs, inhibited papain, too. We conclude that VEGh is a physiological inhibitor of cysteine proteinases and therefore can play a role in the control of inflammatory processes in oral and ocular tissues.

Expression of the gene for tear lipocalin/von Ebner's gland protein in human prostate

Northern analysis of human multiple tissue blots containing poly A + RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes revealed that a prostate specific transcript hybridizes to a tear lipocalin/von Ebner's gland protein (TL/VEGP) gene probe. To characterize this transcript, the corresponding cDNA was amplified by reverse transcription (RT)-PCR. Cloning and sequence analysis showed that it was identical to the tear lipocalin cDNA isolated from human lachrymal glands. Immunohistochemical analysis on thin layer sections of human prostate using a tear lipocalin specific antiserum confirmed the expression of this cDNA in prostate. Thus, our results clearly argue against a unique function of TL/VEGP in human tear fluid or saliva. The human cDNA was expressed in E. coli using the pQE system yielding a recombinant protein which shows biochemical properties identical to the native TL/VEGP.

Localization of substance P NK-1 receptors in rat tongue

In rat tongue, neurons containing substance P terminate in connective tissue, in taste buds, and in lingual epithelium surrounding taste buds in fungiform, foliate and circumvallate papillae. Although many functions have been attributed to these neurons, virtually nothing is known about their physiological function. As a step towards this end, immunocytochemical methods were used to identify the NK-1 receptors (SPR) in rat tongue. SPR-IR was found in the basolateral membranes of taste cells in fungiform, circumvallate and foliate papillae. SPR-IR was not found in the dorsal epithelium or in any structure that could be clearly identified as a neuron. SPR-IR was also found in von Ebner's glands in circumvallate and foliate papillae and in blood vessels in connective tissue in all three papillae. These data suggest that substance P may play a role in taste and/or in oral pain.

Season related ultrastructural features of the parotid, submandibular and von Ebner's glands in hibernating bats.

The parotid, submandibular and the von Ebner's salivary glands of hibernating insectivorous bats (Pipistrellus pipistrellus, Rhinolophus f. e. and Eptesicus serotinus) were studied by transmission electron microscopy and by histochemical methods during various seasons of the year. We report the seasonal ultrastructural changes that occur in the cytoplasmic organelles and the secretory granules of the serous and mucous cells and in the intercalated and striated ducts of the major glands. It previously has been hypothesized that the heterogeneity of the inclusions in the serous granules is closely related to the varying seasonal levels of proteinaceous and glycidic constituents. Lymphatic vessels are present only in the perivascular and periductal connective tissue. Morphofunctional and comparative studies seem to confirm a relationship between secretory activity and nutritional status as well as a likely involvement of the intercalated and striated ducts in determining the composition of the saliva.

Localization of substance P NK-1 receptors in rat tongue

In rat tongue, neurons containing substance P terminate in connective tissue, in taste buds, and in lingual epithelium surrounding taste buds in fungiform, foliate and circumvallate papillae. Although many functions have been attributed to these neurons, virtually nothing is known about their physiological function. As a step towards this end, immunocytochemical methods were used to identify the NK-1 receptors (SPR) in rat tongue. SPR-IR was found in the basolateral membranes of taste cells in fungiform, circumvallate and foliate papillae. SPR-IR was not found in the dorsal epithelium or in any structure that could be clearly identified as a neuron. SPR-IR was also found in von Ebner's glands in circumvallate and foliate papillae and in blood vessels in connective tissue in all three papillae. These data suggest that substance P may play a role in taste and/or in oral pain.

Phagocytic cells in the taste buds of rat circumvallate papillae after denervation.

Phagocytic cells in the taste buds of rat circumvallate papillae after the sectioning of bilateral glossopharyngeal nerves were examined by electron microscopy and immunohistochemistry. Electron micrographs taken 1 day after denervation revealed that flat-shaped cells were present just beneath the taste buds and that their cellular processes extended toward the debris from the degenerating taste buds. At 2-6 days after denervation, long and thin processes of the flat cells surrounded the debris and appeared to have taken them up into the cytoplasm as small vesicles. Evidence for phagocytosis by the flat cells was seen up to 9 days after denervation and again at 24 and 40 days, in correlation to the degeneration and regeneration of the taste buds. Pre-embedding immunohistochemistry using anti-vimentin antibody showed that flat cells strongly reacted with vimentin. Light microscopic immunohistochemistry using anti-macrophage antibodies (ED1, ED2) showed that throughout the post-operative days macrophages were not present underneath or within the taste buds. Most of the ED2-immunoreactive resident macrophages were located in the deep layer of connective tissue, and a few were found in the nerve bundle. ED1-immunoreactive cells were seen in the duct cells of von Ebner's glands and a few were in the trench wall of circumvallate papillae; however, they were also immunoreactive for anti-OX62 antibody, which recognizes dendritic cells. The results indicate that the phagocytic cells of the taste buds are fibroblasts, not macrophages. Moreover, resident macrophages participate in phagocytosis of degenerated nerves together with Schwann cells.

A tumour derived from Ebner's glands: microcystic adnexal carcinoma of the tongue

Microcystic adnexal carcinoma (MAC) is known as an infiltrating but non-metastasizing tumour of the skin, that derives from sweat glands or follicular epithelium. We report on a rare case of MAC of the tongue. The patient had noticed the tumour for two years with slowly increasing dysphagia but no other symptoms of an oropharynx carcinoma. Histological and immunohistochemical analyses showed a similarity between the tumour derived from Ebner's glands of the tongue and MAC of the sweat glands.

EP-GP and the lipocalin VEGh, two different human salivary 20-kDa proteins.

Two salivary 20-kDa proteins [the human lipocalin Von Ebner's gland protein (VEGh) and extraparotid glycoprotein (EP-GP)] show several remarkable similarities and differences. The latter is identical to secretory actin-binding protein (SABP), gross cystic disease fluid protein-15 (GCDFP-15), prolactin-induced protein (PIP), and 17-kDA CD4-binding glycoprotein (gp17). Much is known about the distribution, localization, biochemical characteristics, and molecular biology of these two proteins, yet there are only few clues about their functions.